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  • bob b
    replied
    Tissues Build Firebreaks to Avoid Disease 03/09/2005
    An article in the March 3 issue of Nature1 explains how tissues communicate to fight off infection. As reported before, cells display samples of the proteins they contain on their outer membranes, a process called presentation. Killer T cells wander around, like cops, looking at the presentations. When they recognize alien proteins (antigens), they respond by killing the cell (see 06/27/2003 entry, “Cell to Phagocyte: I’m Dying – Eat Me”).
    Now, Dutch scientists Neijssen et al.2 have found that cells in tissues can also pass these flags to neighboring cells through passageways between them called gap junctions. The uninfected neighboring cells thus signal the cops that a firebreak needs to be constructed to avoid further damage. Australian biologists William Heath and Francis Carbone explain:

    “As well as providing another possible mechanism for initiating immunity by dendritic cells, the gap-junction-mediated cross-presentation described by Neijssen et al. offers an interesting method of efficiently limiting the spread of replicating virus. The authors show that not only will a cell expressing viral proteins be killed by T cells, but so will its closest neighbours – because they present viral peptides obtained through gap junctions. Extending the destruction to adjacent cells may provide a ‘fire-break’ around an infection, ensuring that if low levels of virus have spread to surrounding cells, but have yet to produce sufficient protein to allow recognition, such cells will still be eliminated.”

    The width of the firebreak is controlled, they explain: “The rapid degradation of peptides within the cell’s cytosol means that the spread of peptides through gap junctions will be rather limited, probably allowing the targeting of adjacent cells but not those more than one cell distant from the infection. Thus, the integrity of targeting should be maintained, with only limited bystander destruction.”
    --------------------------------------------------------------------------------
    1Heath and Carbone, “Coupling and cross-presentation, Nature 434, 27 - 28 (03 March 2005); doi:10.1038/434027a
    2Neijssen et al., “Cross-presentation by intercellular peptide transfer through gap junctions,” Nature 434, 83 - 88 (03 March 2005); doi:10.1038/nature03290.

    Bacterial Engineering On Par With Higher Life 03/11/2005
    Bacteria aren’t the simple life-forms microbiologists used to envision, writes Zemer Gitai in Cell.1

    “Recent advances have demonstrated that bacterial cells have an exquisitely organized and dynamic subcellular architecture. Like their eukaryotic counterparts, bacteria employ a full complement of cytoskeletal proteins, localize proteins and DNA to specific subcellular addresses at specific times, and use intercellular signaling to coordinate multicellular events. The striking conceptual and molecular similarities between prokaryotic and eukaryotic cell biology thus make bacteria powerful model systems for studying fundamental cellular questions.”

    This is different from the traditional picture of bacteria, he elaborates:

    “This traditional perspective [of bacteria as fundamentally different from eukaryotes (i.e., simpler] changed significantly in the past decade with dramatic advances in our understanding of bacterial cell biology. Work in multiple species has demonstrated that bacteria are actually highly ordered and dynamic cells. Much like their eukaryotic counterparts, bacterial cells are capable of polarizing, differentiating into different cell types, and signaling to each other to coordinate multicellular actions. The more recent surprises come from advances in fluorescence microscopy, demonstrating that bacterial cells exhibit a high level of intracellular organization. Bacteria dynamically localize proteins, DNA, and lipids to reproducible addresses within the cell and use this dynamic organization to tightly regulate complex cellular events in both space and time.”

    Gitai provides detail on the following examples: (1) Bacteria have homologs of the eukaryotic cytoskeleton, (2) bacterial cells are subcellularly organized (i.e., are not lacking organelles or a nucleus-like function), (3) several mechanisms underlie bacterial subcellular organization, and (4) bacteria are able to engage in multicellular activities.
    “Bacteria are wondrously diverse and resourceful, occupying virtually every environmental niche imaginable,” he writes in conclusion.
    --------------------------------------------------------------------------------
    1Zemer Gitai, “The New Bacterial Cell Biology: Moving Parts and Subcellular Architecture,” Cell, Volume 120, Issue 5, 11 March 2005, Pages 577-586, doi:10.1016/j.cell.2005.02.026.

    Flagellum Described in High-Performance Lingo 04/04/2005
    The bacterial flagellum, a virtual icon of the intelligent design movement, has been studied by many researchers, notably Howard Berg of Harvard, an expert on chemotaxis (the attraction of bacteria to chemical stimuli). Berg was interviewed in Current Biology1 and talked like a race car mechanic when discussing this molecular machine, though he is not involved in the ID movement and believes in evolution. Here are some excerpts:

    • “The modern era [of chemotaxis studies] began in the 1960s with Tetsuo Iino and Sho Asakura in Mishima and Nagoya, who began work on the structure of flagellar filaments (thought then to be primitive bending machines)...
    • the flagellar motor has several pistons and a novel torque-speed relationship....
    • We hope to understand how bacterial chemotaxis works, every nut and bolt. Who would have imagined: receptor complexes that count molecules and make temporal comparisons; activation of a diffusible signal that couples receptors to flagella; reversible rotary engines that drive propellers of variable pitch; force generators, rotors, drive shafts, bushings, and universal joints; a system with prodigious sensitivity, with amplification generated by receptor-receptor interactions? The biggest black box is the motor. We know a great deal about its electromotive and mechanical properties (torque, speed, changes in direction, and so forth) but we do not really know how it works. We need more structural information. This is hard, because essential components are membrane embedded. But even in an age of systems biology, one should not be embarrassed to focus on an isolated network controlling a particular molecular machine.”
    --------------------------------------------------------------------------------
    1Q&A: Howard Berg, Current Biology, Volume 15, Issue 6, 29 March 2005, Pages R189-R190, doi:10.1016/j.cub.2005.03.003.

    Molecular Motors Do Ballet 04/13/2005
    Scientists at University of Illinois studied dynein and kinesin – the tiny molecular trucks that ferry cargo inside the living cell – and found that they are not just individualists: they cooperate in a delicate yet effective performance.
    Some scientists had thought that the two machine types, which travel in opposite directions, were involved in a constant tug-o’war with each other. Instead, reports the university’s news bureau, “The motors cooperate in a delicate choreography of steps.”
    Using high-speed imaging techniques, they determined that “multiple motors can work in concert, producing more than 10 times the speed of individual motors measured outside the cell.” The machines move by “walking” on rails called microtubules in steps 8 billionths of a meter at a time. The team is measuring the force produced by the motion to “further understand these marvelous little machines.” There was no mention of evolution in the report.

    Bacterial Hydrogen Fuel Cell May Yield Cleaner World 04/24/2005
    Scientists at Penn State are working on a new, improved fuel cell. It’s secret? Bacteria that can be coaxed with a little electricity to produce “four times as much hydrogen directly out of biomass than can be generated typically by fermentation alone.” Will you someday be able to harness hydrogen from organic waste to drive your car? Their new electrically-assisted microbial fuel cell can theoretically be used to “obtain high yields of hydrogen from any biodegradable, dissolved, organic matter – human, agricultural or industrial wastewater, for example – and simultaneously clean the wastewater.”

    Genes Must Be Expressed in the Right Order 04/26/2005
    A team of scientists in Switzerland made neural cells switch on a transcription factor earlier during the embryo’s development. The result? Axons (long branches of nerve cells) refused to grow to the spinal cord and to the peripheral target. To the mice, this meant they couldn’t feel things on the skin due to stunted nerves. The paper is published in PLOS Biology. A synopsis of this paper in the same issue (published April 26) explains why the order of expression is important:

    “Building an embryo is like building a house: everything has to be done at the right time and the right place if the plans are to be translated faithfully. On the building site, if the roofer comes along before the bricklayer has finished, the result may be a bungalow instead of a two-story residence. In the embryo, if the neurons, for example, start to make connections prematurely, the resultant animal may lack feeling in its skin.
    On the building site, the project manager passes messages to the subcontractors, and they tell the laborers what to do and where. In the embryo, the expression of specific transcription factors (molecules that tell the cell which DNA sequences to convert into proteins) at different stages of development and in different places controls the orderly construction of the body.”
    --------------------------------------------------------------------------------
    1 Hippenmeyer, Arber et al., “A Developmental Switch in the Response of DRG Neurons to ETS Transcription Factor Signaling,”, [/i]Public Library of Science Biology[/i] Volume 3 | Issue 5 | May 2005, DOI: 10.1371/journal.pbio.0030159.

    World’s Smallest Rotary Motors Coming Into Focus 04/30/2005
    Science April 29 had three articles on the ATP synthase rotary motors that inhabit all living cells.1,2,3 Using creative techniques of extreme microscopy and crystallography, research teams are beginning to get more focused images of the carousel-like rotating engines of both F-type and V-type motors. (V-type enzymes pump ions into the cell to regulate acidity; see 2/24/2003 entry. F-type ATP synthase enzymes produce ATP, the energy currency of the cell; see 09/18/2003 entry.)
    The rotors look like elegant circular rings of helical units arranged at angles to the axis. From the side, they look like “concave barrel with a pronounced waist in the middle, and an inner septum that is probably filled with and electrically sealed by membrane lipids in vivo.” Scientists are still trying to figure out how the ions get into the active-site pockets in the subunits of the ring, and how they create torque to make the carousel go round. It may result from harnessing Brownian motion in a ratcheting manner that only allows rotation in one direction. All the researchers seem surprised that the gear ratio is not an integer, but rather 10:3 in some species, and 11:3 or 14:3 in others; it may be necessary that these motors have a non-integer ratio between the bottom carousel and the top catalytic engine for torque generation and catalytic activity (see 08/10/2004 entry). They are also beginning to understand the nature of the camshaft attached to the carousel that induces ATP production in the top.
    Whatever their mechanism, these little engines, only 12 nanometers tall, are effective. The review by Junge and Nelson says these motors can generate an acidity of pH 2 in lemons and 250 millivolts of electricity in insect guts. We humans also run on electricity. The constant action of quadrillions of these tiny generators running day and night in our bodies keeps all our energy systems humming at about 116 watts (see 02/05/2003 story).
    In another molecular-motor story, Current Biology4 reported about how actin and myosin work during cell division to pinch the two daughter cells apart. David R. Burgess in a review5 states, “Myosin II is the motor for cytokinesis, an event at the end of cell division during which the animal cell uses a contractile ring to pinch itself in half. New and surprising research shows that myosin, either through light chain phosphorylation or through its ATPase activity, also plays an important role in both the assembly and disassembly of the actin contractile ring.”
    --------------------------------------------------------------------------------
    1Wolfgang Junge and Nathan Nelson, “Structural Biology: Nature’s Rotary Electromotors,” Science Vol 308, Issue 5722, 642-644 , 29 April 2005, [DOI: 10.1126/science.1112617].
    2Murata et al., “Structure of the Rotor of the V-Type Na+-ATPase from Enterococcus hirae,” Science, Vol 308, Issue 5722, 654-659, 29 April 2005, [DOI: 10.1126/science.1110064].
    3Meier et al., “Structure of the Rotor Ring of F-Type Na+-ATPase from Ilyobacter tartaricus,” Science, Vol 308, Issue 5722, 659-662 , 29 April 2005, [DOI: 10.1126/science.1111199].
    4E. D. Salmon, “Microtubules: A Ring for the Depolymerization Motor,” Current Biology, Volume 15, Issue 8, 26 April 2005, Pages R299-R302, doi:10.1016/j.cub.2005.04.005.
    5David R. Burgess, “Cytokinesis: New roles for myosin,” Current Biology, Volume 15, Issue 8, 26 April 2005, Pages R310-R311, doi:10.1016/j.cub.2005.04.008.

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  • bob b
    replied
    Cellular UPS Gets Right Packages to Chloroplasts 01/01/2005
    If all your packages were sent correctly over the holidays, consider the job a plant cell has getting 3000 proteins into a chloroplast. Mistakes are not just inconvenient. They can be deadly, or at least bring photosynthesis to a halt. To guarantee proper delivery of components, plant cells have a remarkable shipping system, described in Current Biology by two UK biologists, Paul Javis and Colin Robinson.1 Part of the challenge is getting polypeptides past the double membranes of the chloroplast. A remarkable crew of enzymes and molecular machines puts a shipping label (transit peptide) on each amino acid chain, reads it, routes it to the correct destination, and then removes it:

    “Over 90% of the ~3000 different proteins present in mature chloroplasts are encoded on nuclear DNA and translated in the cytosol [cell fluid outside the nucleus]. These proteins are synthesized in precursor form – each bearing an amino-terminal targeting signal called a transit peptide – and are imported into the organelle by an active, post-translational targeting process (Figure 1). This process is mediated by molecular machines in the outer and inner envelope membranes, referred to as ‘translocon at the outer envelope membrane of chloroplasts’ (Toc) and ‘translocon at the inner envelope membrane of chloroplasts’ (Tic), respectively. Upon arrival in the stroma [chloroplast interior], the transit peptide is removed and the protein either takes on its final conformation or is sorted to one of several internal compartments in a separate targeting process.”

    The authors believe, like most evolutionists, that plastids (including chloroplasts) arose when a primordial cell engulfed another and took over its light-harvesting machinery, a process called endosymbiosis (see 10/01/2004, 09/09/2004, 08/06/2004 and 10/07/2003 headlines). They believe the former cell that became the chloroplast retained only a stripped down version of its genetic code, and most of the DNA instructions for building these 3000 chloroplast proteins got transferred to the nucleus. Yet this means that a tremendous amount of machinery had to be developed to get the proteins to their destinations:

    “Chloroplasts are complex organelles comprising six distinct suborganellar compartments: they have three different membranes (the two envelope membranes and the internal thylakoid membrane), and three discrete aqueous compartments (the intermembrane space of the envelope, the stroma and the thylakoid lumen). One of the consequences of this structural intricacy is that the internal routing of chloroplast proteins is a surprisingly complex process. While envelope proteins may employ variations of the Toc/Tic import pathway to arrive at their final destination, proteins destined for the thylakoid membrane or lumen employ one of four distinct targeting pathways (Figure 1). Thylakoid membrane proteins are targeted by the signal recognition particle (SRP)-dependent and spontaneous insertion pathways, whereas lumenal proteins are targeted by the Sec and Tat pathways....”

    Each of these “pathways” is an assembly-line process involving multiple proteins dedicated to these tasks. Several points brought out in the article make it challenging to perceive of a smooth transition from endosymbiosis to today’s complex shipping and handling pathways (numbering ours):

    1. The transit peptide needs to fit the receptor on the membrane, and another protein has to be ready to cleave it (remove it).
    2. The transit peptides have to be precise to avoid having the protein arrive at the wrong organelle, like the endoplasmic reticulum, mitochondrion or peroxisome – organelles which also accept polypeptides with shipping labels.
    3. Transit peptides are varied. “One might therefore expect chloroplast transit peptides to share well-defined primary or secondary structural motifs,” they say. “On the contrary, transit peptides are remarkable in their heterogeneity. They vary in length from 20 to >100 residues, and have no extended blocks of sequence conservation.”
    4. The transit proteins “do not seem to form secondary structure in aqueous solution” but once they arrive at their target membrane, they seem to take on a characteristic structure.
    5. The polypeptides (precursor proteins) are threaded through the needle of specialized gates in the membrane. There, additional molecular machines (chaperones) make sure they do not fold prematurely.
    6. To get a polypeptide through a membrane involves three steps: contact, docking, and translocation, when the transit peptide is cleaved. This requires energy: a high concentration of ATP must be present for the operation.
    7. The Toc and Tic squads, like a delivery organization with a variety of employees skilled in particular tasks but working on common goals, is made up of multiple proteins, each with its own task to perform, all working in coordination.
    8. Once inside the outer membrane, the polypeptide has to get past the inner membrane. Another set of specialized proteins are available for that task.
    9. A third import apparatus has to complete the task of getting the polypeptide to its final destination. Many go to the thylakoid membrane, rich with light-harvesting structures and ATP synthase (see 08/10/2004 headline).
    10. Those polypeptides bound for the thylakoid membrane have a secondary shipping label (transit peptide). In addition, they may have a “stop-transfer” signal to indicate their destination.
    11. Removal of the secondary transit peptide can occur by “one of two very different pathways,” called Sec and Tat. Sec transports proteins in an unfolded state, but Tat can transport them in a folded state. Each pathway involves multiple proteins working together.
    12. In the Tat pathway, “There is even evidence that some proteins are exported in an oligomeric form” [i.e., several proteins bound together in a complex], “which points to a remarkable translocation mechanism,” they remark. Is this like squeezing a completed sweater through the eye of a needle? “...we currently know very little about this mechanism,” they say. “Somehow, this system must transport a wide variety of globular proteins – some over 100 kDa [kilodaltons] – while preserving the proton motive force and avoiding loss of ions and metabolites.” Their surprise at this indicates it is quite a feat.
    13. The translocation process can expend 30,000 protons, “a substantial cost by any standard.” According to current theory, a pH difference between inner and outer membrane provides the proton flow, but that pH balance must be carefully monitored and regulated.
    14. Another pathway named SRP inserts proteins into the lumen. The authors claim this pathway was “clearly inherited from the cyanobacterial progenitor of the chloroplast,” but admit that there are differences in the insertion pathways and events at the thylakoid membrane in chloroplasts. “...it is fair to state that, while the major players in this pathway have been identified, their modes of action remain unclear and we do not understand how such highly hydrophobic proteins are bound by soluble factors, shuttled to the membrane and then handed over to membrane apparatus and inserted.”
    15. Evolutionists who expected the SRP pathway from E. coli bacteria to act the same in chloroplasts, where homologous proteins were detected, learned otherwise: “Surprisingly, this is not the case. In vitro assays for the insertion of a range of membrane proteins have shown that the vast majority of such proteins do not rely on any of the known protein transport machinery, including SRP, FtsY, Alb3 or the Sec/Tat apparatus, for insertion.” Nor do they rely on nucleoside triphosphates or proton flow.
    16. Speaking of the apparent spontaneous insertion of the thylakoid proteins, they comment, “This unusual pathway for membrane protein insertion appears to be unique to chloroplasts.” Though the typical insertion components are not involved, they believe it would be “overly simplistic” to assume that this pathway requires no “complex insertion apparatus.”
    17. Other pathways than those described above are used for other proteins to get inside the chloroplast. Some are encoded by the chloroplast DNA, translated in the interior, then transported to their destinations.
    18. Chloroplasts have to transport not only the essential light-harvesting proteins, but also “housekeeping” proteins for structural maintenance. These must be imported at their own separate rates depending on the stage of development or the environmental conditions, and have their own specific transit peptides.
    This represents the state of our knowledge on protein transport in chloroplasts. It is only a partial picture of a varied and complicated picture with many players, as their final paragraph makes clear:

    “The Tat pathway manages the remarkable feat of transporting large, folded proteins without collapsing the delta-pH, and we currently know very little about this mechanism. Most membrane proteins use a possibly ‘spontaneous’ insertion mechanism that just does not make sense at the moment – why do these proteins need so little assistance from translocation apparatus, when membrane proteins in other organelles and organisms need so much? And how do these thylakoid proteins avoid inserting into the wrong membrane? We have gone some way toward understanding the rationale for the existence of all these pathways, but the thylakoid may still have surprises in store.”

    By contrast, another paper in the same issue of Current Biology2 makes confident claims that the endosymbiosis theory has been demonstrated with diatoms (see 10/01/2004 and 07/21/2004 headlines about diatoms). They suggest that it was dangerous for genes to remain in the plastids, because of free radicals generated by the photosynthesis machinery, and because of higher mutation rates, and that’s why most of them wandered to the nucleus.
    --------------------------------------------------------------------------------
    1Paul Jarvis and Colin Robinson, “Mechanisms of Protein Import and Routing in Chloroplasts,” Current Biology, Volume 14, Issue 24, 29 December 2004, Pages R1064-R1077, doi:10.1016/j.cub.2004.11.049.
    2Nisbet, Killian and McFadden, “Diatom Genomics: Genetic Acquisitions and Mergers,” Current Biology Volume 14, Issue 24, 29 December 2004, Pages R1048-R1050, doi:10.1016/j.cub.2004.11.043.

    DNA Translators Cannot Tolerate Editor Layoffs 01/12/2005
    We’ve explained elsewhere about the family of molecular machines called aminoacyl-tRNA synthetases (see 05/26/2004 entry and its embedded links). Their job is to associate each word of DNA code (codon) with its corresponding piece of a protein (amino acid). In a very real sense, they translate the DNA code into the protein code. One amazing capability of these machines is that they proofread their work. They can differentiate between similar molecules, and edit out incorrect pieces inserted by mistake. Scientists from Scripps Institute writing in PNAS1 thought they would watch what happened when they gave one of these translators a mutation that diminished this editing ability. It wasn’t pretty:

    “The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain. A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids. Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase. Ambiguity retarded cell growth at most temperatures in rich and minimal media. These growth rate differences were seen regardless of the carbon source. Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function. Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors. Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.”

    In short, removing the editing created big problems. The poor bacteria were stunted and vulnerable to malfunctions. When the translator could not maintain high fidelity by editing out mistakes, crippled proteins were produced, and the organism became a sitting duck for the harsh realities of survival.

    Update 01/26/2005: This paper generated a commentary in PNAS by Randall Hughes and Andrew Ellington of the University of Texas.2 They agreed that “over the long run, there has been and will continue to be tremendous selective pressure to maintain the current genetic code.” But they surmise that, since not all the substituted amino acids produced fatalities, evolution might take advantage of them. “Taking advantage of protein misfolding might at first seem to be an improbable event,” they admit, “but this phenomenon is conceptually similar to other ways in which organisms take evolutionary advantage [sic] of even inclement environments.” Like citizens under siege scrounging for food, they envision a cell under stress with “a general need to explore a larger genetic space or a larger protein folding space or both.” Maybe the cell has already planned for such things through experience. “To the extent that organisms have encountered environmental stress intermittently over evolutionary time,” they write, “it may even be advantageous to establish some sort of regulatory feedback between stress and phenotypic exploration.” In the end, though, they agree that the cell works hard to prevent such errors and possesses exquisite means to eliminate typos. That means it will be difficult to find ways to change the genetic code in lab organisms: “simple substitutions will be an uphill battle.”
    --------------------------------------------------------------------------------
    1Bacher, Crécy-Lagard and Schimmel, “Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0409064102, published online before print January 12, 2005.
    2Randall A. Hughes and Andrew D. Ellington, “Mistakes in translation don’t translate into termination,” Proceedings of the National Academy of Sciences USA, February 1, 2005, vol. 102, no. 5, pp. 1273-1274.

    Simple Darwinian Theories Have to Be Abandoned 01/17/2005
    Mutate one gene and a cascade of changes can result. This effect is called pleiotropy (see 10/01/2003 entry). According to an article by Stephen Strauss reporting for the Canadian newspaper Globe and Mail, “The emerging richness of pleiotropy means that any simple Darwinian notion of what is going on during natural selection has to be abandoned.”
    Unless Darwinians can show that the positive changes outnumber the negative effects, pleiotropy seems to spell difficulty, if not doom, for neo-Darwinian theory, which relies on beneficial mutations. But if beneficial mutations are rare to begin with, how can evolutionary theory face the new problem of pleiotropy? “The simplest answer,” Strauss writes, “is that nearly 150 years after Darwin first explained the theory of evolution, the richness of multiple effects from the same gene is such that existence itself seems problematic”.
    Strauss gives examples of a few more nuanced proposals for salvaging Darwinian evolution: “Faced with what amounts to a growing daily confusion of genetic effects, biologists are proposing new and more highly refined theories of evolution.” Some biologists hope that some mutations have only minor effects. Others are looking for examples of single mutations that might have a cascade of good effects. He ends on a hopeful note: “With modern genetics increasing the supply of data about the multiple functions of genes, evolutionary biologists are increasingly confident that they are going to be able to do what Darwin promised but couldn’t quite delivery [sic] -- truly explain the origin of species.”

    Ribosome Unties the Messenger-RNA Gordian Knot 01/19/2005
    Cells needing to translate their DNA into proteins have a problem. The messenger RNAs, the molecules that carry the genetic code from the nucleus to the translating machine called the ribosome, get tied up in knots. How does the ribosome untie them before they can begin translating? Takyar et al., writing in Cell,1 explored this problem and found that the ribosome has a novel solution.
    If you have seen the film Unlocking the Mystery of Life, you watched a messenger RNA molecule, nice and straight, exit the nuclear pore complex and neatly enter the ribosome, like a man reclining in a barber chair waiting to get a haircut. Unfortunately, things are not so simple. Because of chemical affinities between the bases of the RNA molecule, the bases attract other bases (base-pairing) or else fold over on themselves, forming amorphous lumps (secondary structure). Untangling this mess would be like straightening out a chain of several hundred magnets that has clumped together.
    The untangling problem is not unique to messenger RNA (mRNA). DNA in the nucleus also has to be unwound. Each of the processes of “replication, DNA repair, recombination, transcription, pre-mRNA splicing, and translation” have their own specialized enzymes, called helicases, that latch onto the nucleic acids and work their way down the helix, unwinding them for whatever subsequent operation is necessary. Until now, though, no helicase was found associated with the ribosome. It turns out the helicase activity is built-in.
    The ribosome has an entry tunnel and exit tunnel. As the mRNA strand enters, specialized proteins named S3, S4 and S5 are precisely placed to form a ring around the mRNA helix. They grab the phosphate groups on the side chains and separate the base pairs.2 There’s only room in the tunnel for a single strand. As the interior of the ribosome pulls the mRNA through, this entry-tunnel helicase, built into the walls of the tunnel, effectively “melts” the double strands, sending in a clean single strand for the translation machinery to work on. And how does the ribosome pull it in?

    “In their studies of ratcheting of the two ribosomal subunits between the pre- and posttranslocation states, Frank and Agrawal (2000) observed a reciprocal expansion and contraction in the diameter of the upstream and downstream tunnels, suggesting that these two features may alternately grab and release the mRNA during translocation of mRNA. This dynamic behavior in the downstream tunnel could also be related to its helicase function.”

    The action seems analogous to those old Dymo labelmakers people used to use for labeling household items. You remember: as your hand clicked the machine, the tape came in one tunnel and out another. In the case of the ribosome, the entry and exit tunnels alternately expand and contract, forcing the mRNA molecule to ratchet through the system. The ratchet prevents backward motion and also is delicate enough to prevent breakage of the single strand during the unwinding process.
    The placement of S3, S4 and S5 in the tunnel is critical. The researchers found that when they were mutated, the helicase activity stopped. Because it latches onto the phosphates, which are universal to RNA molecules, they can unwind any strand, regardless of the sequence of base pairs.
    The authors do not speculate on how this helicase system, which is unique to the ribosome, evolved. They only note that if it did, the unwinding puzzle needed to be solved by the very first living cell:

    “The inescapable presence of secondary structure within mRNA coding sequences must have been one of the first problems encountered in the transition from an RNA world to a protein world [sic] and may have resulted in coupling of ribosomal helicase activity with the fundamental mechanics of translocation.”

    How this was accomplished by a sequence of random changes, they do not explain.
    --------------------------------------------------------------------------------
    1Takyar et al., “mRNA Helicase Activity of the Ribosome,” Cell, Vol 120, 49-58, 14 January 2005.
    2It was not clear to the authors whether the helicase pulls the bases apart with the expenditure of energy. It may be that the helicase can take advantage of spontaneous separation. Base pairs tend to “breathe” as their weak hydrogen bonds stretch. The helicase may be able to latch onto the nucleotide during its spontaneous separation, as if saying “Aha! Gotcha!” and prevent the hydrogen bond from re-forming.

    Design Paper Published in PNAS 01/26/2005
    A team of Japanese and American biologists, from Caltech and University of California and elsewhere, describe the heat shock response in the cell. They not only compare this biological system to good engineering, but treat the engineering paradigm as a proper approach to the study of cellular systems: in fact, they say, “Viewed from this perspective, heat shock itself constitutes an integral functional module. Such a characterization of functional modules is extremely useful, because it provides an inventory list of cellular processes. An analogy would be a list of machines and their function in a factory.” For more design language, look at the abstract:

    “Molecular biology studies the cause-and-effect relationships among microscopic processes initiated by individual molecules within a cell and observes their macroscopic phenotypic effects on cells and organisms. These studies provide a wealth of information about the underlying networks and pathways responsible for the basic functionality and robustness of biological systems. At the same time, these studies create exciting opportunities for the development of quantitative and predictive models that connect the mechanism to its phenotype then examine various modular structures and the range of their dynamical behavior. The use of such models enables a deeper understanding of the design principles underlying biological organization and makes their reverse engineering and manipulation both possible and tractable. The heat shock response presents an interesting mechanism where such an endeavor is possible. Using a model of heat shock, we extract the design motifs in the system and justify their existence in terms of various performance objectives. We also offer a modular decomposition that parallels that of traditional engineering control architectures.”

    The paper is filled with design words: engineering, robustness, feedback loops, feed-forward loops, modularity, performance, functional criteria, and the like – all but the buzzphrase “intelligent design.” For example, “Biology and engineering share many similarities at the system level, including the use of complexity to achieve robustness and performance rather than for minimal functionality.”
    The only mention2 of biological evolution is a passing reference in the final discussion that, in the surrounding design language, seems almost irrelevant: “The formulation of such a problem aside, the physical implementation of any of its solutions seems to have been evolutionarily solved by using a number of recurring motifs...” How it was solved, and who solved it, is left unexplained. Instead, the authors seem enthusiastic that a design-theoretic approach, viewing cellular mechanisms the way a computer scientist would reverse-engineer software, can be a fruitful avenue for research:

    “However, to understand the operational principles of a certain machine, to repair it, or to optimize its performance, it is often necessary to consider a modular decomposition of the machine itself. Such a decomposition does not necessarily require stripping the machine down to the component level but rather identifying its submodules with their predefined functionalities. “A particularly successful such modular decomposition has been extensively used in the field of control and dynamical systems, where components of a system are classified in terms of their role with respect to the regulation objective. Similar decompositions exist in computer science, for example, because modularity is a basic principle of good programming.”

    The authors make no mention of a Programmer, or state their personal beliefs about origins. But that, again, supports a principle stated frequently in the intelligent design literature: the identity of the designer is not the issue. Design detection is a purely scientific question, and the design-theoretic approach is a fruitful avenue of research.
    --------------------------------------------------------------------------------
    1El-Samad, Kurata, Doyle, Gross and Khammash, “Surviving heat shock: Control strategies for robustness and performance,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403510102, published online before print January 24, 2005.
    2The only other possible allusion states, “Indeed, in higher level languages, a complicated programming task is usually divided into a set of modules, subroutines, or objects, with simple well defined interfaces. This results in flexible and robust programs, whose modules can be designed almost separately and, as such, are more easily evolvable.” However, being in the context of computer program design, the statement implies guided evolution – i.e., upgrading – by intelligent design, not evolution by an undirected or Darwinian process.

    Your Motors Are Turbo-Charged 01/30/2005
    Think how fast 6000 rpm is. It would redline on most cars. Yet you have motors in your body that make that speed look like slow-mo.
    The Japanese have taken great interest in the cellular machine ATP synthase since its rotary operation was discovered in 1996 (see 12/22/2003 entry). Maybe it’s because they like rotary engines. ATP synthase is an essential protein complex that generates ATP (adenosine triphosphate), the energy currency of the cell. Found in the membranes of mitochondria and chloroplasts, it runs on an electrical current of protons, from sunlight (in plants) or digestion (in animals). It is a reversible engine: it can just as easily generate protons from the dissociation of ATP. It has five major protein parts, including a rotor, a stator, and a camshaft. The F0 domain runs like a waterwheel on protons and turns the camshaft. Three pairs of lobes in the F1 domain catalyze ATP from ADP and phosphate, in a three-phase cycle of input, catalysis, and output. Each revolution generates 3 ATP.
    Hiroshi Ueno and team, reporting in PNAS,1 have invented new techniques for studying and measuring the tiny motors. Now, with the aid of a high-speed camera running at 8,000 frames per second, they have clocked the rotational speed of the entire F0F1-ATP Synthase motor at 352 revolutions per second, a whopping 21,120 rpm.
    Although this molecular machine exists in all life forms, they used motors from a thermophilic bacterium. To monitor the action, the team fastened a microscopic bead to the carousel of c subunits. At 25° C, it ran at 230 rps. At 45° C, it ran at 650 rps. Extrapolating up to 60° C, the organism’s optimum growth temperature, they speculate that it could be running as fast as 1,600 rps – an unbelievable 96,000 rpm – and that with nearly no friction and almost ideal efficiency. While they caution that reservation is needed whether these “enormous numbers” are actually achieved, they do say with confidence that the rotation rates they measured are much higher than earlier claims. “It is intriguing to learn,” they say, “whether these rapid rotations are really occurring in living cells.”
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    1Ueno et al., “ATP-driven stepwise rotation of F0F1-ATP synthase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0407857102, published online before print January 24, 2005.

    Genes Evolving Downward 02/02/2005
    Those assuming the evolution of eukaryotic genomes has progressed upward in complexity may find the following abstract from PNAS1 startling:

    “We use the pattern of intron conservation in 684 groups of orthologs from seven fully sequenced eukaryotic genomes to provide maximum likelihood estimates of the number of introns present in the same orthologs in various eukaryotic ancestors. We find: (i) intron density in the plant-animal ancestor [sic] was high, perhaps two-thirds that of humans and three times that of Drosophila; and (ii) intron density in the ancestral bilateran [sic] was also high, equaling that of humans and four times that of Drosophila. We further find that modern introns are generally very old, with two-thirds of modern bilateran introns dating to the ancestral bilateran [sic] and two-fifths of modern plant, animal, and fungus introns dating to the plant-animal ancestor [sic]. Intron losses outnumber gains over a large range of eukaryotic lineages. These results show that early eukaryotic gene structures were very complex, and that simplification, not embellishment, has dominated subsequent evolution.”

    In their paper, Harvard biologists Scott Roy and Walter Gilbert used the maximum-likelihood phylogenetic method instead of maximum parsimony, and feel it provided a better ancestral tree. In fact, they used the same data as other scientists who used parsimony, and got very different results. They are emphatic about their conclusions:

    “These results push back the origin of very introndense genome structures over a billion years to the plant-animal split. Indeed, ancestors at the divergences between major eukaryotic kingdoms as well as the ancestral bilateran appear to have harbored nearly as many introns as the most intron-dense modern organisms. This is a sharp repudiation of the common assumption that intron-riddled gene structures arose only recently.
    In addition, our analysis shows that the majority of introns are themselves very old. Two-thirds of bilateran introns were present in the bilateran ancestor ; 40% of opisthokont introns were present in the opisthokont ancestor; and 40% of plant, animal, and fungal introns were present in the plant-animal ancestor. This is quite different from what is commonly assumed and surprising in light of relatively fast rates of intron turnover observed in nematodes and flies.”

    This bias toward intron loss instead of gain appears to be a general trend among eukaryotes, they conclude. What does this mean? The only way to rescue an evolution toward “improvement” with these results is to suggest that introns are bad, like parasites, and that over time, eukaryotes got better at ridding themselves of them. They reject that and other notions, assuming instead that “It seems much more likely that different selection or mutation regimes for introns along different lineages are driving the observed instances of gene streamlining.” Although intron function and evolution is still largely unknown, they leave only an admission of ignorance of what their results mean – only that geneticists had better re-examine their assumptions:

    “These results contradict the assumption that genome complexity has increased through evolution. Instead, species have repeatedly abandoned complex gene structures for simpler ones, questioning the purpose and value of intricate gene structures. These results suggest a reconsideration of the genomics of eukaryotic emergence.”
    --------------------------------------------------------------------------------
    1Scott W. Roy and Walter Gilbert, “Complex early genes,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0408355101, published online before print February 1, 2005.

    Molecular Machine Parts Stockpiled in Readiness for Assembly 02/06/2005
    A team from the European Molecular Biology Laboratory has done a “4D” time-and-materials study of molecular machines, analyzing the process of assembly, reports EurekAlert. They found that the cell stockpiles some parts and holds them in storage, but adds the crucial elements just in time.

    “The researchers discovered that in yeast, key components needed to create a machine are produced ahead of time, and kept in stock. When a new machine is needed, a few crucial last pieces are synthesized and then the apparatus is assembled. Holding off on the last components enables the cell to prevent building machines at the wrong times. That’s a different scenario from what happens in bacteria, which usually start production of all the parts, from scratch, whenever they want to get something done.
    “We saw a clear pattern as to how the complexes are assembled,” says Søren Brunak from DTU. It’s unusual to find such concrete patterns in biology, compared to physics for example, due to the evolutionary forces that change living systems. But using this new model, the underlying principle became very clear.”

    The authors next want to find out how long components stay around after use. Their results were published in Science1 Feb. 4; see also the brief on EurekAlert.
    --------------------------------------------------------------------------------
    1Lichtenberg et al., “Dynamic Complex Formation During the Yeast Cell Cycle,” Science, Vol 307, Issue 5710, 724-727, 4 February 2005, [DOI: 10.1126/science.1105103].

    Survival of the Fittest – or the Luckiest? 02/06/2005
    Evolutionists assume that bacteria spread because they evolve resistance to antibiotics and become more fit to survive. That’s apparently not true, says a story in EurekAlert about a study from Imperial College, London: the spread of bacteria appears to be due to chance alone.
    Here are two quotes from the article by team members explaining the finding:

    “Dr Christophe Fraser, from Imperial College London, a Royal Society University Research Fellow and one of the authors, says: “Microbiologists have assumed for some time that some disease strains spread more successfully than others. In fact we found that the variation in the communities we studied could be explained by chance. This was surprising, especially considering all the potential advantages one pathogen can have over another, such as antibiotic resistance and differences in host immunity.”
    Dr Bill Hanage, from Imperial College London, and also one of the authors, says: “When we look at a sample and see that some strains are much more common than others, it’s tempting to think that there must be something special about them. In fact, they could just be the lucky ones, and that’s what it looks like here. Most of the variation in the spread of these pathogens can be explained by chance alone.”

    The team studied three pathogenic bacteria and followed the social patterns of the humans they infected. There was no clear association between success at spreading and fitness for spreading .
    A related commentary by Dan Ferber in Science1 had another surprise about bacteria: they are not immortal. Reproducing strains in a culture apparently show their age. What does this mean? For one thing, the results “make it unlikely that natural selection produced an immortal organism.” For another, “It’s one of those exciting results that makes you take a fresh look at what you think you know.” One observer is not sure the populations that stopped growing were aging; maybe they were taking a break for repairs. But another said the new findings “put the onus of proof on anyone who claims that cells can be immortal.”
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    1Dan Ferber, “Immortality Dies as Bacteria Show Their Age,” Science, Vol 307, Issue 5710, 656 , 4 February 2005, [DOI: 10.1126/science.307.5710.656a].

    Introns Engineered for Genetic Repair 02/18/2005
    Scientists at Purdue University are using bacterial machines to treat cancer and other diseases. These machines, called Group I introns, were thought to be useless:

    “Once thought of as genetic junk, introns are bits of DNA that can activate their own removal from RNA, which translates DNA’s directions for gene behavior. Introns then splice the RNA back together. Scientists are just learning whether many DNA sequences previously believed to have no function actually may play specialized roles in cell behavior.”

    Though the function of introns is still mysterious (see 02/02/2005 entry), they appear to be highly conserved in both archaea and eukarya, suggesting they are important. Bacteria have Group I introns that do self-splicing. Eukaryotes have Group II introns that are spliced by one of the most complex molecular machines in cells, the spliceosome (see 09/17/2004 entry).

    Clutch Enables Your Motors to Achieve 100% Efficiency 02/23/2005
    Those little ATP synthase motors (see 01/30/2005 entry) in your body and (in all living cells) made news again in Nature1 last week. Scientists in Tokyo performed an ingenious set of experiments to measure the efficiency of the F1 synthesizing domain. They attached a tiny magnet to the camshaft so that they could turn it with electromagnets at will, and they carefully measured the amount of ATP synthesized or hydrolyzed as the motor turned anticlockwise or clockwise under their control. In the hydrolysis cycle, they found that the motor did not waste ATP; each molecule was successfully hydrolyzed with perfect efficiency, to the limits of their detection.
    A particular focus of their investigation was the role of the eta subunit, which is attached to the gamma camshaft. During hydrolysis, the “downhill” function, it did not seem to matter whether eta was present or absent. But in the “uphill” process (synthesizing ATP), it made a dramatic difference. Without eta, each rotation produced, on average, only one product, but with it, they got three per revolution, with at least 77% efficiency. The actual efficiency was probably higher, but was hard to measure for such small entities. In best cases, it was 100%, they said: “Therefore our data point to an excellent mechanochemical coupling efficiency. In the best cases, we observed the postulated value of three ATPs synthesized per turn.”
    “These results are consistent with the ubiquity of this strategic enzyme that fuels most of the energy consuming biological processes,” they said. “The present work reveals the unexpected importance of the eta-subunit in the synthesis of ATP.” Though its precise function remains to be discovered, it was known to play a regulatory role; now, this team suspects it acts like a structural switch or clutch to lock the enzyme into synthesis mode. Without it, the tiny motor undergoes wasteful slippage.

    As a reminder to recent readers, you can find a wonderful animation of this molecular machine on the website of German biochemist Wolfgang Junge. It is labeled “F0F1-ATPSynthase (animation)” See also his Model Schematic.
    --------------------------------------------------------------------------------
    1Rondelez et al., “Highly coupled ATP synthesis by F1-ATPase single molecules,” Nature 433, 773 - 777 (17 February 2005); doi:10.1038/nature03277
    Last edited by bob b; October 27, 2006, 02:56 PM.

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    Is the Evolution of Bacterial Resistance a Just-So Story? 09/12/2004
    Evolutionists frequently point to the emergence of bacterial resistance to antibiotics as an example of Darwinian evolution occurring right under our noses. Bruce R. Levin of Emory University, writing in the Sept. 10 issue of Science,1 is not so sure about that. He points out that cells might just have a built-in mechanism to shut down growth and reproduction in times of stress (the SOS response), to minimize the damage from toxins in the environment. He points to two studies in the same issue that indicate how noninherited resistance to antibiotics can be generated without reference to Darwinian natural selection.
    What’s more interesting in his report is his rebuke against fellow Darwinists who leap to unsubstantiated tales of evolution to explain how these mechanisms come about. His final paragraph states:

    “It is easy to concoct just-so stories to explain the evolution of a mechanism that, like the SOS response, produces quiescent cells that are refractory to lethal agents. Yet it seems unlikely that ampicillin was the original selective force [sic] responsible for the evolution [sic] of the induction mechanism observed by Miller and colleagues. A bigger challenge to those in the evolution business is to account for the generation of lower fitness cell types when they do not provide an advantage to the collective, like the persisters of Balaban et al. in the absence of antibiotics. Then again, just like people, bacteria do some seemingly perverse things that are not easy to account for by simple stories of adaptive evolution.

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    1Bruce R. Levin, “Microbiology: Noninherited Resistance to Antibiotics,” Science, Vol 305, Issue 5690, 1578-1579, 10 September 2004, [DOI: 10.1126/science.1103077].

    Peering Into Paley’s Black Box: The Gears of the Biological Clock 09/15/2004
    William Paley’s famous “watchmaker argument” for the existence of a Designer, though intuitively logical to many, has been criticized by naturalists on the grounds that one cannot compare mechanical devices to biological ones. Biological “contrivances” might operate on totally different principles than mechanical ones made by humans we know.
    Michael Behe’s 1996 book Darwin’s Black Box was built on the theme that, until recently, the living cell was a “black box” to biologists: i.e., a system whose inner workings lay hidden from us. But now with the rapid advances in molecular biology, we are finding the cell to be a complex factory of molecular machines.
    These themes of Paley and Behe seemingly converge in a commentary by Susan S. Golden (Texas A&M) in PNAS about biological clocks.1 Golden works at the Center for Research on Biological Clocks in the Texas A&M Biology Department, and was struck by recent findings in two other papers in PNAS on the circadian rhythms of “primitive” blue-green algae (cyanobacteria). To her, they suggested we are opening the black box of biological clocks, and finding treasures that look remarkably familiar to the clocks we know:

    “A physiological black box is to a biologist what an ornately decorated package is to a small child: a mysterious treasure that promises delightful toys within. With fitting elan, a small community of scientists has ripped open the packaging of the cyanobacterial circadian clock, compiled the parts list, examined the gears, and begun to piece together the mechanism. Over the past 2 years, the 3D molecular structures have been solved for the core components of the cyanobacterial circadian clock: KaiA, KaiB, and KaiC. In a surprisingly literal analogy to mechanical timepieces, the protein that seems to be at the heart of the clock mechanism, KaiC, forms a hexameric ring that even looks like a cog: the escape wheel, perhaps. Previous work has shown that KaiC has an autophosphorylation activity, and that the presence of KaiA and KaiB modulates the extent to which KaiC is phosphorylated. In this issue of PNAS, Nishiwaki et al. biochemically identify two amino acid residues on KaiC to which phosphoryl groups covalently attach, and show the necessity in vivo of a phosphorylation-competent residue at these positions. By searching the crystal structure for evidence of phosphorylated sites, Xu et al. pinpoint a third residue that may “borrow” the phosphoryl group dynamically. Together, their work contributes richly to our understanding of what makes the gears mesh and turn to crank out a 24-h timing circuit....
    Because each of these components (at minimum) is a dimer [composite of two molecular chains], KaiC is known to be a hexamer [composite of six chains], and other proteins may be present as well, the cyanobacterial clock can be thought of as an organelle unto itself: a “periodosome” that assembles and disassembles during the course of a day, defining the circadian period.”

    The term “periodosome” means “time-keeping body” – i.e., clock. Her diagram shows KaiC as a six-sided carousel to which phosphate groups and other subunits attach and detach during the diurnal cycle. The feedback between the units provides the periodicity of the clock, similar to the back-and-forth pendulum in a grandfather clock or the escape wheel in a wristwatch. How is the clock tuned to the day-night cycle? Where do the parts come together, and how do the clock gears mesh with other cellular machines? We don’t know yet; the box has just been opened.
    The clocks examined in these papers are the “simple” clocks of blue-green algae, compared to the much more complex biological clocks in eukaryotes. Even about these relatively simple systems in cyanobacteria much remains to be understood, but our initial glimpses into the inner workings of a biological clock at the molecular level remind her of the delight of opening a chest of toys for the first time:

    “Identification of other potential components of the periodosome, intracellular localization of the clock parts, and elucidation of other potential modifications all may yield gears that are required to smoothly tick away the time and ensure that daughter cells do not run fast or slow.
    The cyanobacterial clock box, no longer black, is a chest filled with bioluminescence and attractive toys. Putting together the pieces to design a clock is a tedious task, but S. elongatus is a gracious host, and the guests at the party are hard at work.”

    --------------------------------------------------------------------------------
    1Susan S. Golden, “Meshing the gears of the cyanobacterial circadian clock,“ Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0405623101

    Secrets of the Spliceosome Revealed 09/17/2004
    A husband and wife team from Hebrew University has revealed the structure of the spliceosome, one of the most complex molecular machines in the cell (see 09/12/2002 headline), in more detail than ever before, says EurekAlert. The spliceosome is responsible for cutting out the introns in messenger RNA after it has transcribed DNA, and also for “alternative splicing” that rearranges the exons to produce a variety of proteins from the same DNA template: “Alternative splicing, which underlies the huge diversity of proteins in the body by allowing segments of the genetic code to be strung together in different ways, takes place in the spliceosome as well.”
    The Sperlings found a tunnel between the two major subunits of the machine where they believe the cutting and splicing operations take place, and also a cavity that might provide a safe haven for the messenger RNA strand, like a waiting room, before its surgery. Also, they found that four spliceosomes are bound together into a “supraspliceosome” which is able to do “simultaneous multiple interactions, rather than by a stepwise assembly” as inferred from other experiments in vitro. Their investigation in vivo (within a functioning, living cell) revealed even more complexity in the composite machine than had been seen in the individual machines:

    “Such a large number of interactions that the cell has to deal with can be regulated within the supraspliceosome. Having the native spliceosomes as the building blocks of this large macromolecular assembly, this large number of interactions can be compartmentalized into each intron that is being processed. At the same time, the whole supraspliceosome enables the communication between the native spliceosomes, which is needed for regulated splicing. The organization of the supraspliceosome, like other macromolecular assemblies that exist as preformed entities, avoids the necessity to recruit the multitude of splicing components each time the spliceosome turns over. In that sense, the overall coordination of the cellular interactions is reduced from the hard work of repeatedly placing each piece in the correct position of the puzzle to the relatively simpler work of coordinating the preformed puzzle.”

    In short, “The supraspliceosome represents a stand-alone complete macromolecular machine capable of performing splicing of every pre-mRNA independent of its length or number of introns.” They found that the individual spliceosomes are joined with a flexible joint like a hinge to provide flexible interactions and communication. Their work was published in Molecular Cell Sept. 10.1
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    1Sperling et al., “Three-Dimensional Structure of the Native Spliceosome by Cryo-Electron Microscopy,” Molecular Cell, Volume 15, Issue 5, 10 September 2004, Pages 833-839; doi:10.1016/j.molcel.2004.07.022.

    Bacterial Flagellum Reveals New Structural Complexity 10/27/2004
    The bacterial flagellum, the unofficial mascot of the Intelligent Design movement, got more praise from the evolutionary journal Nature this week: Samatey et al.1 analyzed the hook region in detail and found that it is composed of 120 copies of a specialized protein that “reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.”
    Christopher Surridge, commenting on this paper in the same issue,2 adds that this joint must be able to bend up to 90 degrees in a millisecond or less while rotating at up to 300 times per second. He says that the researchers describe “how they determined the atomic structure of this super-flexible universal joint, and thereby how it achieves such a feat of engineering.”
    --------------------------------------------------------------------------------
    1Samatey et al., “Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism,” Nature 431, 1062 - 1068 (28 October 2004); doi:10.1038/nature02997.
    2Christopher Surridge, “Molecular motors: Smooth coupling in Salmonella,” Nature 431, 1047 (28 October 2004); doi:10.1038/4311047b.

    [b]“Crucial Evolutionary Link” Found for Eukaryotes 11/05/2004
    Often the opening words of a news story are what stick in the memory: “crucial evolutionary link.” The corroborating evidence, however, is buried in technical details of the press release from Rockefeller University, posted on NewsWise. In short, the researchers claim:

    “Scientists believe the emergence of organelles, compartments in the eukaryotic cell’s cytoplasm that perform such functions as energy production, waste removal and protein synthesis, and a nucleus evolved between 2 and 3 billion years ago.
    One hypothesis regarding the evolution of eukaryotic cells suggests that the endomembrane system developed because some ancient bacterial cells had the ability to sharply curve their membranes, allowing them to form internal membrane structures as well as to engulf other organisms. The findings reported by [Michael P.] Rout and colleagues [Rockefeller University] suggest that an ancestor of an NPC component, called the Nup84 complex, may have been a key molecular sculptor responsible for such a reshaping of the membrane.”

    To find out what the Nup84 complex is, you have to wade through the boring body of the article. For one thing, Nup84 is complicated:

    “...the scientists ... found that the Nup84 complex in yeast is composed of two types of protein structures, “alpha solenoids” and “beta propellers.” Two of the proteins are beta propellers, three are alpha solenoids and two are composed of beta propeller “heads” attached to alpha solenoid “tails.” The scientists showed that the architecture of the Nup84 complex also appears in the NPCs of human and plant cells and is therefore conserved throughout eukaryotes.”

    As our regular readers know, any functional protein is composed of a chain of amino acids, all left-handed, assembled by a complex factory of molecular machines (see online book). The function of a protein is dependent on the precise sequence of the amino acids and the way the chain is folded with the help of other machines named chaperones. When you have a complex of proteins working together (and most proteins work in complexes), the requirements for specified complexity are even higher. The authors are assuming that this protein complex Nup84 emerged through a Darwinian process.
    What’s the gist of the missing link claim? Basically, that Nup84 not only can curve a membrane, it is also involved in shuttling cargo around the cell. Since both prokaryotes and eukaryotes do that, but only eukaryotes curve their membranes to form organelles, they concluded that Nup84 is a missing link, a “crucial evolutionary link.”

    Bacterial Hypodermic Needle Examined 11/10/2004
    Those who have seen the film Unlocking the Mystery of Life might recall seeing the image of the “needle-nosed cellular pump” that some evolutionists claim was an intermediate for the bacterial flagellum. Those wishing to investigate this claim further might want to see the renditions that a Yale team produced of the pump, called a Type III Secretion System (TTSS), in the Nov. 5 issue of Science.1 Their introduction describes the machine:

    “TTSSs are composed of more than 20 proteins, including a highly conserved group of integral membrane proteins, a family of customized cytoplasmic chaperones, and several accessory proteins, placing TTSSs among the most complex protein secretion systems known.”

    Their images of the TTSS show parts resembling exquisitely crafted rings, gears, sockets, rods and tubes. The parts are flexible and undergo drastic conformational changes during assembly that amount to reprogramming of the parts. Here’s a small sample of what transpires during the assembly of this one molecular complex:

    “Contoured longitudinal sections revealed conformational changes that occurred during the transition from the base to the fully assembled needle complex (Fig. 3, A and B). The cuplike protrusion that emerged from the basal plate of IR1 moved down, while an inward, clamping movement of IR2 redefined the shape of the cavity that is located below the basal plate of the base (movie S2). These conformational changes may provide the structural basis for the functional reprogramming of the TTSS machinery, which upon completion of needle assembly, switches from secreting the needle protein PrgI, the inner-rod protein PrgJ (see below), and the regulatory protein InvJ ... to secreting the effector proteins that are delivered into the host cell. On the opposite side of the basal plate, the socketlike structure underwent an outward movement, which created an attachment point for the inner rod (movie S2). A similar outward movement was observed for OR1, which created space for the needle to dock at the outermost perimeter of the base (movie S2). These changes were complemented by an outward movement of OR2 and a drastic remodeling that flattened the septum, sealing the apical side of the base, against OR2 during needle assembly (Fig. 3, A and B; movie S2). This rearrangement of the septum is essential for creation of the secretion channel and transformed part of InvG from being a barrier into forming two scaffolds that enable assembly of the needle and the inner rod. Like the socket structure at the basal end of the chamber, these new scaffolds likely serve as adaptors, accommodating the symmetry mismatches between the base, the needle, and the inner rod.”

    Thus, the assembly of the TTSS involves not only parts coming together, but a coordinated series of shape changes of the parts relative to one another such that they fit together tightly, to enable the finished pumping action. We know the TTSS largely from “virulence of many Gramnegative bacteria pathogenic for animals and plants”.
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    1Marlovits et al., “Structural Insights into the Assembly of the Type III Secretion Needle Complex,&148; Science, Vol 306, Issue 5698, 1040-1042, 5 November 2004, [DOI: 10.1126/science.1102610].

    Flagellar Oars Beat Like Galley Slaves In Synchronization 12/26/2004
    The Dec. 14 issue of Current Biology1 investigated another mystery in the operation of eukaryotic flagella:

    “Flagella are microtubule-based structures that propel cells through the surrounding fluid. The internal structure of a flagellum consists of nine parallel doublet microtubules arranged around a central pair of singlet microtubules (Figure 1). Force for propulsion is provided by thousands of dynein motors anchored in rows along one side of each doublet, which can walk along the microtubule of the adjacent doublet. In order to produce coordinated bending of the flagellum, these dynein motors — organized into multi-headed complexes called the inner and outer dynein arms — must produce their power strokes in synchrony, like the oarsmen on an ancient Mediterranean war-galley. But whereas oar-strokes were coordinated by a continuous drum-beat, it is much less clear how flagellar dynein motors are synchronized.”

    The authors of the paper consider growing evidence that the central microtubule pair provides the drumbeat, with the aid of “a protein complex called the dynein regulatory complex, located between the spokes and the dynein arms.” However, “The molecular mechanism by which the central pair regulates dynein is not known.”
    --------------------------------------------------------------------------------
    1Kimberly A. Wemmer and Wallace F. Marshall, “Flagellar Motility: All Pull Together,” Current Biology Volume 14, Issue 23, 14 December 2004, Pages R992-R993, doi:10.1016/j.cub.2004.11.019.

    Cells Find Signal in the Noise 12/20/2004
    Parents at an amusement park know the challenge of picking out their child’s voice, or even hearing their own hollering, in the noise of the crowd. Yelling won’t help much if the rest of the crowd is yelling also. Acoustic engineers know that raising the volume while playing back a noisy tape amplifies the noise as well as the signal. Cells have a novel way of meeting this challenge, as two Japanese mathematical biologists discuss in PNAS.1 Cells are continuously sending and receiving chemical messages, a process called signal transduction. Treating the cell signal transduction network like a physical system of receivers and amplifiers, the researchers noted that a cell, like an amusement park, is an intrinsically noisy place, yet some of the reactions are very sensitive. “How cells respond properly to noisy signals by using noisy molecular networks is an important problem in elucidating the underlying ‘design principle’ of cellular systems,” they say in the introduction. How do the sensitive reactions get their messages through all that noise?

    “Because intracellular processes are inherently noisy, stochastic reactions process noisy signals in cellular signal transduction. One essential feature of biological signal transduction systems is the amplification of small changes in input signals. However, small random changes in the input signals could also be amplified, and the transduction reaction can also generate noise. Here, we show theoretically how the abrupt response of ultrasensitive signal-transduction reactions results in the generation of large inherent noise and the high amplification of input noise. The inherently generated noise propagates with amplification through intracellular molecular network. We discuss how the contribution of such transmitted noise can be shown experimentally. Our results imply that the switch-like behavior of signal transduction could be limited by noise; however, high amplification reaction could be advantageous to generate large noise, which would be essential to maintain behavioral variability.”

    They categorized the noise as intrinsic, coming from the reaction itself, to extrinsic, coming from other reactions. This is somewhat like hearing your own voice vs. the yelling of those around you. The intrinsic noise has higher frequency than the extrinsic noise. As one source of noise becomes dominant, it reaches a crossover point where the other source is less dominant. This provides a kind of signal, or switch, which the cell can use to advantage:

    “From our result, it can be further suggested that if the extrinsic noise dominates, the upstream reactions affect the fluctuation of the most downstream reaction, which determines the cellular behavior. As a result, the behavioral fluctuations are made up of the contributions of the fluctuations of several upstream reactions. On the other hand, if the intrinsic noise dominates, only the intrinsic noise of the most downstream reaction determines the behavioral fluctuations. As a result, the behavior could be simpler than the case in which extrinsic noise is dominant....
    ....Consequently, the low-frequency modulations in the downstream reactions can be affected by the behaviors of upstream reactions, whereas the high-frequency modulations are expected to be independent of upstream reactions.”

    As a result, a bacterium can respond to chemicals in the environment, the hemoglobin in your blood can respond to changing conditions in the capillaries, genes can respond correctly to requests for expression, and complex cascades of cellular reactions can respond to the signal from any reaction in the series, in the midst of all the noise. “Therefore,” they conclude, “the result implies that the extrinsic noise is essential to maintain the behavioral variability in wild-type bacteria.” Their experiments related to three relatively simple reactions, and their analysis considered primarily linear response. Many cellular reactions involve nonlinear behavior. “In these cases,” they admit, “the relation between the response and the fluctuations can be more complicated than the relations we studied.”
    --------------------------------------------------------------------------------
    1Tatsuo Shibata and Koichi Fujimoto, “Noisy signal amplification in ultrasensitive signal transduction,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403350102, published online before print December 29, 2004.

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    Cell Technology Celebrated 07/01/2004
    Humans are just beginning to imitate the manufacturing techniques cells use all the time, right under our noses. A book just came out about the subject, entitled Bionanotechnology: Lessons from Nature by David S. Goodsell. It’s hard to tell if Christof M. Niemeyer was more impressed with the book or with the living machines themselves, in his review in the July 1 issue of Nature.1 He writes,

    “Nanotechnology is perfectly realized in biological systems. Cells are essentially biological assemblers that build thousands of custom-designed molecules and construct new assemblers. In Bionanotechnology, structural biologist David Goodsell describes what biology can teach us about engineering and manufacturing at the nanometre scale.”

    “Small wonder,” reads a caption whimsically; “antibodies ... are just one example of the way nature uses nanotechnology.” Niemeyer mentions a few more examples of “the composition and structural principles of biomolecules harnessed in the cell” :

    • the machinery of DNA transcription and translation
    • biomolecular motors
    • the information-driven synthesis of biological molecules
    • the energetics and regulation of biological processes
    • the traffic across membranes and signal transduction along them
    • the interplay of myosin and actin filaments within the muscle sarcomere

    Although the book focuses on how humans can tinker with this biological nanotechnology, reviewer Niemeyer enjoyed every page of this “fascinating journey.”
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    1Christof M. Niemeyer, “Living Machinery,” Nature 430, 20 (01 July 2004); doi:10.1038/430020a.

    Archaea Have Their Own Proofreading Mechanism 07/07/2004
    A team of Yale biochemists investigated a proofreading mechanism in one-celled organisms from the domain Archaea and found it different, but just as effective, as its counterpart in domains Bacteria and Eukarya (the latter including all plants and humans). Their work was published online in PNAS July 6.1
    The particular instance involved the ability to discriminate between two similar amino acids, threonine and serine, on the molecule that connects the amino acid to the transfer RNA (aminoacyl-tRNA synthetase, or aaRS). Members of Archaea have an enzyme that bears no sequence similarity, but is “functionally conserved” (i.e., does the same thing), to that of the other domains. The archaeal gene is “unrelated to, and absent from,” bacterial and eukaryotic genomes. The authors term this an instance of “functional convergence of unrelated domains” that “assures specificity” of the correct amino acid to the tRNA molecule. This “appears to be the first aaRS found to use two evolutionarily unrelated editing domains,” they state. “The functional convergence between the two ThrRS editing domains is highlighted by the observation that both depend on an absolutely conserved set of histidine residues for their function.”
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    1Korencic et al., “A freestanding proofreading domain is required for protein synthesis quality control in Archaea,” Proceedings of the National Academy of Sciences USA , 10.1073/pnas.0403926101.

    Cell Cargo Speeds On Bidirectional Highways 07/12/2004
    As reported here numerous times (e.g., 06/14/2004, 12/04/2003, 04/14/2003, 03/28/2003, 02/25/2003, 12/17/2002, 09/26/2002, 03/26/2002, 02/01/2002, 12/06/2001, 08/17/2001, 06/19/2001, 02/21/2001), cells have an elaborate interstate highway system with molecular trucks hauling cargo back and forth. Scientists have known that the cellular highways have polarities labeled plus and minus, and that molecular motors typically go one way. Some motors, like kinesin, drive only in the plus direction, while others, like dynein, go in the minus direction. Now, it is becoming apparent that most pieces of cargo have at least one of each kind of motor, with a stickshift that allows it to drive in forward or reverse. The state of our knowledge about bidirectional transport is explored by Michael Welte in the July 13 issue of Current Biology.1
    Welte examines the evidence that many, maybe all, moving cargoes have bidirectional ability. In the microscope, certain organelles like mitochondria and melanosomes are seen to move back and forth rapidly, eventually making it to their target. Why is this, and how is it done? Does the organelle grab motors out of the cytoplasm? Are both motors working in a tug-o’war? Welte cites evidence against these possibilities, and suggests (although hard evidence needs to be found), that the cargo carries both motors, and a “complex coordination machinery ... ensures that when one motor is actively engaged with the microtubule, the other motor is turned off.” Moreover, this coordination machinery, whatever it is, may be under the influence of regulatory enzymes. “If the coordination machinery can attach to cargo independent of the motors,” he surmises, “distinct variants of the coordination machinery could be targeted to different cargoes, thus allowing cargo-specific coordination and regulation.”
    It seems odd, though, that cargoes would undergo a back-and-forth random walk instead of making a beeline to the target. Welte figures there must be biological justification for this behavior, so he examines some possibilities:

    Economy: “If cargoes always carry motors for both directions, net transport can easily be adjusted or even reversed by simply tweaking the relative activity of the two motors. This is likely to be much quicker than assembling a new set of motors on a cargo, and also allows transport to be abruptly altered depending on cellular needs. It even makes it possible to tune the overall speed of transport by altering the relative contribution of trips in the non-dominant direction.”
    Setting Up Polarized Distributions: “Sometimes it is necessary to set up a distribution rather than to confine the organelles to a single point .... Even if cargoes accumulate at a certain point (e.g. near plus-ends when motion is biased in the plus-end direction), trips in the non-dominant direction will tend to spread the cargoes out along the tracks, away from the point of accumulation. Modeling shows that by altering the relative contributions of plus- and minus-end trips, a wide range of steep to flat steady-state distributions can be achieved.”
    Avoiding Obstacles and Exploring Space: “As cytoplasmic dynein often steps sidewise to adjacent proto-filaments, a bidirectional cargo could find itself on the opposite side of the microtubule even after a short minus-end excursion. If it now switches back to kinesin I, it can pass the obstacle. Bidirectionally moving cargoes should, therefore, be less likely to contribute to disastrous traffic jams .... The random walk of bidirectional cargoes allows a single cargo to explore a large region of cellular space, especially if tracks are disordered.”
    Error Correction: “During unidirectional transport, the critical event that determines directionality of motion is the attachment to either a plus- or minus-end motor. A wrong attachment will cause misdelivery of the cargo. During bidirectional transport, the net direction of transport is determined by the balance of plus- and minus-end trips and can, therefore, be continually evaluated and even altered if physiological conditions change. Thus, bidirectional transport may facilitate error correction.

    It must be remembered that these motors are operating in the dark without eyes, like automated railroad cars. They don’t have sentient drivers on radios, but rather respond to chemical signals in the environment. Apparently these behaviors achieve the best solution to many complex problems. “Bidirectional transport by opposite-polarity microtubule motors is just one example of multiple motors working together to achieve carefully choreographed transport,” Welte says, as he concludes with a list of open problems needing further elucidation.
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    1Michael A. Welte, “Bidirectional Transport along Microtubules,” Current Biology, Volume 14, Issue 13, 13 July 2004, Pages R525-R537, http://dx.doi.org/10.1016/j.cub.2004.06.045.

    Plant “Evolutionary Leftover” Now Deemed Vital 07/22/2004
    Photorespiration, “a biological process in plants, thought to be useless and even wasteful” and “just an evolutionary leftover” from an age when carbon dioxide was more prevalent, has been found to be “necessary for healthy plant growth and if impaired could inhibit plant growth,” according to a UC Davis study published in PNAS. (see also summary on EurekAlert). It functions as a way to inhibit nitrate assimilation. Some agricultural scientists assumed it was an unnecessary process to be genetically engineered out of plants because it was wasteful, “But the new UC Davis study suggests that there is more to photorespiration than meets the eye and any attempts to minimize its activity in crop plants would be ill advised.”

    Spaghetti in a Basketball: How the Cell Packs DNA for Controlled Access 07/28/2004
    The beginning sentence of an article in Current Biology1 can’t help but grab your attention:

    “Imagine trying to stuff about 10,000 miles of spaghetti inside a basketball. Then, if that was not difficult enough, attempt to find a unique one inch segment of pasta from the middle of this mess, or try to duplicate, untangle and separate individual strings to opposite ends. This simple analogy illustrates some of the daunting tasks associated with the transcription, repair and replication of the nearly 2 meters of DNA that is packaged into the confines of a tiny eukaryotic nucleus. The solution to each of these problems lies in the assembly of the eukaryotic genome into chromatin, a structural polymer that not only solves the basic packaging problem, but also provides a dynamic platform that controls all DNA-mediated processes within the nucleus.”

    The article by Craig L. Peterson and Marc-André Laniel is otherwise boringly titled “Histones and histone modifications,” but after this appetizing start, goes into detail about how the tangled mess of alphabetized pasta is exquisitely controlled, folded, unfolded and copied continuously inside the cell, with the help of numerous protein and RNA parts.
    Of special importance are the histone proteins that comprise chromatin. Scientists have been discovering for several years now that these histones have “tails” of amino acids that can be altered through numerous ways. These alterations, called “post-translational modifications,” seem to influence the DNA wrapped around them in many important ways. They signal genes to activate for transcription, places needing DNA repair, places to start or repress DNA elongation or replication, where to silence telomeres, places to deposit more chromatin, and more. A table in the article lists 95 histone modifications and their functions that are known so far. Some are involved in mitosis (cell division), spermatogenesis, X-chromosome inactivation (silencing one of the two X-chromosomes in the female), apoptosis (programmed cell death), DNA “memory” and other important cell processes. Some have said these modifications constitute a “histone code” (see “Cell memory borders on the miraculous,” 11/04/2002 headline). These authors term it differently, but no less amazing: “rather than a histone code there are instead clear patterns of histone marks that can be differentially interpreted by cellular factors, depending on the gene being studied and the cellular context.” Activities like DNA repair or replication are often accompanied by histone modifications, for instance, as if one enzyme leaves its mark on a histone to signal a follow-up function. Complexes of small RNAs and enzymes depend on these markers to know where to go and what to do; the histone tails serve as attachment points for specific enzymes. And if that is were not amazing enough, the interplay of neighboring histone markers, or cross-talk, can have “a profound effect on enzyme activity.” The authors explain, “Thus, in many ways histone tails can be viewed as complex protein-protein interaction surfaces that are regulated by numerous post-translational modifications. Furthermore, it is clear that the overall constellation of proteins bound to each tail plays a primary role in dictating the biological functions of that chromatin domain.” Finally, since some of these histone states can survive cell division, they augment what’s inherited beyond DNA alone. The authors provide no suggestions on how this system might have evolved.
    On a related subject, three geneticists from Scotland describe, in the same issue of Current Biology,2 how DNA packs itself so tightly and efficiently. There are specialized proteins called condensins that perform this job. They are members of a set of hairpin-shaped enzymes called “structural maintenance of chromosomes” enzymes (SMCs, see 08/07/2002 headline). The authors remind us that “These extraordinary molecules are conserved [i.e., unevolved] from bacteria to humans.” Scientists are beginning to be able to watch condensin do its amazing work in real time (see “DNA folds with molecular velcro,” 06/07/2004 headline). Condensin produces “supercoils” of DNA, one of many steps in packing the delicate DNA strands into a hierarchy of coils that results in a densely-packed chromosome. “It is not entirely clear how the DNA is held in this supercoiled state,” they say, “but several studies suggest that the V-shaped arms of the condensin complex may loop and clamp the DNA in place.” This clamping is “rapid and reversible.” Scientists watching the process in both bacteria and humans are “showing that both vertebrate and bacterial condensins drive DNA compaction in an ATP-dependent fashion with a surprising level of co-operativity that was not fully appreciated.” The condensin molecules work as a team; if not enough condensin is around, nothing happens.
    These authors point out also that condensin is just one of many enzymes involved in chromosome formation. Think about how remarkable it is that during each cell division, the chromosomes are structured so reliably that they can be labeled and numbered under the microscope. “Our own proteomic analysis,” they claim, “has identified over 350 chromosome-associated proteins, so there is clearly more work to be done.”
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    1Peterson and Laniel, “Histones and Histone Modifications,” Current Biology, Volume 14, Issue 14, 27 July 2004, Pages R546-R551, doi:10.1016/j.cub.2004.07.007.
    2Porter, Khoudoli and Swedlow, “Chromosome Condensation: DNA Compaction in Real Time,” Current Biology, Volume 14, Issue 14, 27 July 2004, Pages R554-R556, doi:10.1016/j.cub.2004.07.009.

    Gymnastic Enzyme Acts Like Logic Gate 07/31/2004
    An enzyme named vinculin undergoes “drastic” conformational changes, reports William A. Weis in the July 29 issue of Nature.1 Vinculin, with over a thousand amino acid links, is important at membrane junctions for transporting materials in and out of the cell. It helps cellular “glue” exit the membrane so that neighboring cells can adhere to one another, such as in epithelial tissues.
    Weis reports on recent studies that show vinculin undergoes radical conformational changes during its action. It will only build the adhesive junction when the necessary components are in place. Nothing happens unless the participants are ready; “the binding energy of several partners is needed to overcome the thermodynamic and perhaps kinetic barriers to activation,” he says. “Viewed in this way, vinculin functions as a logical AND gate, in which binding of two partners is required to generate an output, in this case a stable multi-protein complex”. What’s more, this automatic regulation is essential for its function; it prevents inappropriate assembly if the amount of product is unstable.
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    1William A. Weis, “Cell biology: How to build a cell junction,” Nature 430, 513 - 515 (29 July 2004); doi:10.1038/430513a.

    Cell Nucleus Complexity Baffles Evolutionists 08/06/2004
    In her inimitable way, Science reporter Elizabeth Pennisi has once again portrayed a scientific controversy undergoing active ferment. This time it’s about the evolutionary origin of cell nuclei, which she terms “specialized, DNA-filled command centers.”1 At the conclusion, she gives prominence to a “provocative, but circumstantial and controversial” suggestion that viruses taught cells how to wrap their DNA in double membranes with controlled access. Since the idea presupposes that viruses preceded all three domains of life – prokarya, eukarya and archaea – “If this is true, then we are all basically descended from viruses,” as a believer puts it. The idea is unpalatable to some. “I do not believe [it],” a German molecular biologist retorts. “The idea of the viruses ‘inventing’ [eukaryotic cells] from scratch is hard for me to conceive.”
    Pennisi treats the new viral theory as tentative at best. What’s more revealing in her article are the problems with previously-popular ideas, and why. According to her, the key insight at a meeting in France last month on the subject was: “They had underestimated the complexity of the eukaryotic cell’s 1.5-billion-year-old [sic] precursor. The data presented indicated that this ancestral cell had more genes, more structures, and more diverse biochemical processes than previously imagined”. For a glimpse why, look at Pennisi’s brief description of the nucleus:

    “Each nucleus in a eukaryotic cell consists of a double lipid-based membrane punctuated by thousands of sophisticated protein complexes called nuclear pores, which control molecular traffic in and out of the organelle. Inside, polymerases and other specialized enzymes transfer DNA’s protein-coding message to RNA. Other proteins modify the strands of RNA to ensure that they bring an accurate message to the ribosomes outside the nucleus. The nucleus also contains a nucleolus, a tightly packed jumble of RNA and proteins that are modified and shipped out of the nucleus to build ribosomes.”
    (For more on the nuclear pore complex, see 06/17/2002 and 01/18/2002 headlines.)

    Eukaryotes are distinguished from bacteria by their double-membrane nuclei. “The nuclear distinction between prokaryotes and eukaryotes shaped early speculation about the development of complex life,” Pennisi says about ideas floating around up to the 1970s. Some thought eukaryotes were evolved prokaryotes, and others thought prokaryotes were degenerate eukaryotes. But then Carl Woese created new woes by identifying bacteria-like cells that were distinct from both prokaryotes and eukaryotes: so different, in fact, to warrant classification in their own domain – archaea. Others soon were surprised to find that eukaryotes appeared to have genes from both bacteria and archaea.
    So another story was born, the endosymbiont or merger hypothesis. This proposed that eukaryotes arose from “the ancient symbiotic partnership between bacteria and archaea.” That theory came under fire from the discovery of faint but distinct nuclei in an unusual group of bacteria, named planctomycetes, that live in soil and fresh water. Some of these planctomycetes have organelles and double-membraned sacs of DNA and RNA. According to a critic of the merger model, these observations “turn the dogma that ‘prokaryotes have no internal membranes’ upside down” Now, it seems no one is sure which way is up.
    There’s more to cause vertigo for evolutionists: the complexity of the nuclear pore complexes (NPCs). “Explaining these structures has always posed a sticking point for nuclear evolution [sic].” For one thing, “without pores, the nucleus can’t function.” But for another thing, Pennisi continues, the same planctomycetes, and possibly some other archaea and prokaryotes, apparently possess structures resembling these complex traffic-control gates. “Bacteria with nuclear pores and internal membranes, features typically considered eukaryote-specific, suggest that the nucleus was born much earlier than traditionally thought.”
    For some, that leaves as the leading contender the controversial theory that viruses first invented the nucleus. This, however, only pushes the complexity of nuclei and their pores farther back in time, and foists a huge design problem on earth’s most primitive biological entities. That is why the molecular biologist quoted earlier can’t believe that simple viruses created such complex structures from scratch. Pennisi shares a few speculations, based on circumstantial evidence, how it might have happened. But when she ends by pushing the answer to the future, it underscores the fact that no current theory accounts for the origin of the nucleus:

    “Did a virus provide the first nucleus? Or was it something an early bacterial cell evolved, either on its own or in partnership with an archaeum? To resolve the origin of the nucleus, evolutionary biologists are exploring new techniques that enable them to determine relationships of microorganisms that go much further back in time.... “

    The biologists in France argued and discussed many ideas. “But when it came to accounting for how the nucleus was born,” Pennisi admits, “no single hypothesis bubbled to the top.” She quotes French molecular biologist Patrick Forterre who said, “It’s like a puzzle. People try to put all the pieces together, but we don’t know who is right or if there is still some crucial piece of information missing.”
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    1Elizabeth Pennisi, “Evolutionary Biology: The Birth of the Nucleus,” Science, Vol 305, Issue 5685, 766-768, 6 August 2004, [DOI: 10.1126/science.305.5685.766].

    ATP Synthase: Another Unexpected Case of Fine Tuning 08/10/2004
    ATP synthase, the miniature rotary motor that powers our cells, has been a subject of great interest since the elucidation of its rotary function won three scientists a Nobel prize in 1997. As an example of a precision-crafted, true electric rotary motor in living systems (another being the larger bacterial flagellum), it also provides a classic case study in intelligent design vs. evolution. It has been the subject of frequent updates in these pages (start at 02/13/2004 and work backwards). Now, another discovery about this ATP-synthesizing engine has revealed a deeper level of fine tuning. Japanese scientists publishing in PNAS1 found a precision coupling between two components that was unexpected, yet apparently essential.
    For review, recall that ATP synthase has two functional domains, named F0 and F1. The F1 part that actually synthesizes ATP from ADP + P is now fairly well understood. It is composed of three pairs of lobes that spring-load ATP with every 120o turn of the camshaft, each pair of lobes either loading, catalyzing or ejecting an ATP molecule. The F0 domain, however, has been harder to study. Scientists knew it looks like a carousel of identical proteins, labeled c subunits. Linked to it is a camshaft, named the gamma subunit, that drives the synthesis of ATP in F1. Scientists knew the F0 carousel runs on protons delivered by a gumball-like mechanism named the a subunit (see 12/22/2003 headline). But up till now, they were not sure how many c subunits comprised the carousel – or even if the number mattered. Some studies had hinted that the F0 motor contained anywhere from 8 to 13 c subunits, depending on the species. Now, the team of Mitome et al. found the answer: it is 10, and it must be 10 and only 10. Other numbers don’t work. That’s strange. It means that F0 needs 10 protons per revolution, but F1 produces 3 ATP per revolution. The ratio 10:3 is not an integer. How can that be?
    The scientists arrived at the number 10 by customizing F0 rings with fixed numbers of c subunits, 2 through 14. Then they linked them to the F1 domains and watched how much ATP was synthesized. Results were obtained for only c=2, 5, and 10, which is interesting, considering that 2 and 5 are factors of 10. The c=2 and c=5 cases produced a little ATP, and c=10 produced the maximum. All the other numbers produced none. The team deduced, therefore, that 10 (or one of its factors) is essential to match the proton-loading mechanism of the a subunit.
    The scientists also measured the proton flow through their custom carousels when disengaged from F1 and found, again, that 10 was the only number that worked. Without 10 c subunits, no protons flowed. Divide a circle of 360o by 10, and you get a 36o angle per c subunit during a complete revolution of the F0 motor. The F1 domain, by contrast, produces ATP for each 120o turn, or 3 ATP per complete revolution. The scientists seemed surprised that the proton-ATP ratio, “one of the most important parameters in bioenergetics,” is not an integer. It’s as if three protons are sufficient to generate an ATP sometimes and four other times, because one cannot have a third of a proton. Wouldn’t it be more logical if the number of c subunits was a multiple of three, say 6, 9, or 12? With c=9, for instance, the camshaft angle would regularly line up with the F1 lobes every 3 protons, yielding one ATP every time, nice and neat. The fact that it does not means that the coupling between F0 and F1 is not strict, as with toothed gears, but “permissive” – as if the two domains rotate according to their own structural needs, and are coupled together by a adaptor mechanism that has some degree of freedom to either twist or slip.
    The scientists ruled out slippage. They knew that the camshaft can only produce an ATP in the F1 domain when it is lined up perfectly at the 120o steps. Instead, they found that there is enough elastic flexibility in the camshaft to permit twist up to 40o during its rotation. This flexibility allows the two domains to work separately, each according to its optimum configuration, with the twisting camshaft able to rock back and forth a little to give the F1 lobes time to complete their work. In scientific lingo, “The flexibility of gamma allows both the F0-gamma and F1-gamma interfaces at the free-energy minima to stay in conformations adequate for the proton transport in F0 and the catalysis in F1 despite the step-size mismatch, providing sufficient time for those events to take place.”
    One more thing. There isn’t much tolerance for error in this system. The team found that a single point mutation at a spot named E56 in the c subunit was enough to quench all proton flow and all ATP synthesis: “This result provides evidence that each of all 10 E56 in the c-ring is indispensable.” Also, the quantity of 10 subunits in the c-ring is critical, because 8, 9, 11, 12 and other numbers did not fit the gumball proton-delivery system of the a subunit: “Thus, the proton transport through F0 requires very strict arrangement of contact surface between F0-a and F0-c in the F0 assembly and even a rotary displacement as tiny as 3.3o (360o / 10 – 360o / 11) seems to be enough to disable a proton transfer between them.”
    The team made their measurements on ATP synthase motors from a species of thermophilic (heat-loving) bacteria. They feel they have found a coupling strategy in living systems that could demonstrate a general principle: “Here, we report the permissive nature of the coupling between proton transport and ATP synthesis of F0-F1, but such nature of the coupling can be general among other biological motor systems to connect critical well tuned microscopic events in the large domain motions.”
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    1Mitome et al., “Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403545101, published online 8/09/04.

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    Virus: Like DNA in a Hard Plastic Shell 05/07/2004
    A European team of biophysicists studied the mechanical properties of a virus and found the shell, made of protein, to act like hard plastic. Writing in PNAS,1 they described the coat of a bacteriophage they studied:

    “The protective proteinaceous shells (capsids) of viruses are striking examples of biological materials engineering. These highly regular, self-assembled, nanometer-sized containers are minimalistic in design, but they combine complex passive and active functions. Besides chemical protection, they are involved in the selective packing and the injection of the viral genetic material.”

    The capsids look like oblong, geometric shapes with pointy ends. The DNA is packed inside under pressure, and the coat can withstand indentations of 30%. “The measured Young’s modulus,” they found, “is comparable with that of hard plastic.” They seemed to admire the little cases: the bacteriophage capsid is :

    “remarkably dynamic yet resilient and tough enough to easily withstand the known packing pressure of DNA (~60 atmospheres). These capsids, thus, not only provide a chemical shield but also significant mechanical protection for their genetic contents. Viral shells are a remarkable example of nature’s solution to a challenging materials engineering problem: they self-assemble to form strong shells of precisely defined geometry by using a minimum amount of different proteins.”

    The team is looking at these miniaturized packages for inspiration in the burgeoning field of nanotechnology.
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    1Ivanovska et al., “Bacteriophage capsids: Tough nanoshells with complex elastic properties,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0308198101, published online before print May 7, 2004.

    Botulinum Toxin Deactivated by One Slight Change 05/10/2004
    A researcher at Brookhaven National Laboratory mutated a botulinum enzyme by just one amino acid, and abolished its toxicity. The mutation, a change from a glutamate to a glutamine at one position, increased the distance from a zinc atom to a water molecule by 0.6 angstrom, less than one tenth of a billionth of a meter. This was enough to prevent the botulinum enzyme from cleaving its target protein, a neurotransmitter. The modified enzyme could still bind to it, but not cleave it.

    Former “Junk DNA” Now Considered Essential 05/10/2004
    The term “junk DNA” seems to be fading with each new discovery. Helen Pearson, reporting for [i\Nature Science Update[/i], leads with the line “‘Junk’ DNA reveals vital role: Inscrutable genetic sequences seem indispensable.” They don’t know what it does yet, but the assumption is it must be important for evolution to hang onto it for so long. Pearson writes,

    “If you thought we had explored all the important parts of our genome, think again. Scientists are puzzling over a collection of mystery DNA segments that seem to be essential to the survival of virtually all vertebrates. But their function is completely unknown.
    The segments, dubbed ‘ultraconserved elements’, lie in the large parts of the genome that do not code for any protein. Their presence adds to growing evidence that the importance of these areas, often dismissed as junk DNA, could be much more fundamental than anyone suspected.”

    Researchers found 480 sequences that are identical between humans, mice and rats, and “largely match up with chicken, dog and fish sequences too,” but do not exist in invertebrates such as sea squirts and fruit flies.
    Scientists can only guess what these sequences do. One idea is that they “control the activity of indispensable genes.” Another is that they may slice and splice RNA into different forms. Or perhaps they may control embryo growth. Pearson describes the initial reactions to the discovery that junk DNA is not junk after all:

    To solve the conundrum, experts predict a flurry of studies into the enigmatic DNA chunks. “People will be intrigued by this [finding],” says Kelly Frazer who studies genomics at Perlegen Sciences in Mountain View, California. “It is the kind of stuff that blows people away.”
    She quotes one researcher who said, “It absolutely knocked me off my chair.” It was hard to believe these sections could be 100% identical. Some thought they must have contaminated their samples. “The presence of exact copies in different animals suggests that even tiny changes in the sequence of these segments destroy whatever they do,” Pearson surmises, “and have been weeded out during evolution” [sic] whereas other parts have been free to accumulate mutations.
    Clearly there is a lot of work ahead, Pearson says. Finding the function of the ultraconserved elements is just the tip of the iceberg. There are other vast tracts of similar so-called “junk DNA” whose functions await discovery.

    On a related subject, Current Biology has news on introns (see 09/03/2003 headline). A dispatch by Arlin Stoltzfus begins, “The evolutionary origin of spliceosomal introns remains elusive. The startling success of a new way of predicting intron sites suggests that the splicing machinery determines where introns are added to genes.” New techniques show the splicing sites are not random, because observers can predict where they will be found with uncanny accuracy. The “putative benefits” of introns that “justify their existence” are still unknown. Apparently, the cell has “mechanisms of targeted intron gain.”
    See also the May 12 BBC News report on this finding.

    Male Imparts More to Embryo than Just DNA 05/12/2004
    A team of biologists have confirmed that male sperm RNAs are delivered to the oocyte along with the DNA. Specifically, paternal messenger RNAs are delivered to the egg. These might influence development and put the male’s imprint on the developing zygote. Writing in Nature,1 the researchers speculate what the finding means:

    “Why should spermatozoa messenger RNAs be transferred to the oocyte? Messenger RNAs encoding proteins that bind nucleic acids, such as protamine-2, are likely to be deleterious and are probably degraded following entry, and a similar fate may await other RNAs that gain access. But some may have a role in the developing zygote: for example, clusterin (also known as sulphated glycoprotein-2, or SGP-2) is delivered to the oocyte and has been implicated in cell-cell and cell-substratum interactions, enhancement of fertility rate, lipid transportation, membrane recycling, stabilization of stress proteins, and promotion or inhibition of apoptosis. These may therefore be required in the early zygote but unnecessary in the oocyte. Alternatively (or in addition), these and other unidentified molecules, such as small interfering RNAs (siRNAs), may participate in processes such as pronuclear formation, the orchestration of events leading to oocyte activation, the transition from maternal to embryonic gene control, and the establishment of imprints in early embryos.”

    But haven’t cloning and parthenogenesis experiments shown the male contribution to the zygote is dispensable?

    “However, the success of such experiments and of somatic-cell nuclear transfer is limited, as is the production of human embryonic stem cells after somatic-cell nuclear transfer. This may be because sperm RNAs contribute to early development. Transcripts that are specific to male germ cells play a role in the differentiation of embryonic stem cells and their function may not be easily replaced.”

    They conclude that these accessory RNAs delivered in sperm may be necessary for fertility, and may influence the developing embryo with a signature only the male can provide.
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    1Ostermeier et al., “Reproductive biology: Delivering spermatozoan RNA to the oocyte,” Nature 429, 154 (13 May 2004); doi:10.1038/429154a.

    Cell Requires Two Keys to Let Cargo Pass 05/13/2004
    For high-security environments, guards sometimes require two independent authentication methods. Before humans came up with this trick, the cells in their bodies were already using it. Itoh and Camilli explain in the May 13 issue of Nature:1

    “Our cells contain a series of distinct compartments that do different jobs and have different properties. The membranes that clad each of these compartments – like the plasma membrane that encases the cell – are defined by precise molecular compositions, which are preserved despite the continuous influx and efflux of components in transit to and from other cellular locations. Precision is the hallmark of this flow of traffic, too, which must be directed appropriately between compartments. All of this is achieved, in part, by the reversible recruitment of regulatory proteins from other parts of the cell to specific membranes or membrane regions. A growing amount of evidence hints that membrane lipids cooperate with membrane proteins to control this recruitment.”

    They refer to work by Godi et al. in Nature Cell Biology that shows a dual-key authentication mechanism in the cell. With “at least two independent, but synergistic, mechanisms,” cargo is only allowed to bind to a membrane if it binds correctly to two cytosolic proteins. This can be envisioned as a kind of code:

    “The interaction of cytosolic proteins with both lipids and proteins on a target membrane is an efficient dual-key strategy to control their recruitment to membranes. Only when both the lipid-binding and protein-binding sites are engaged is the interaction with the membrane strong enough. The two elements of the code can be controlled independently, affording the possibility of fine-tuning the spatial and temporal regulation of recruitment.”

    Itoh and Camilli provide no suggestions on how such a system might have evolved.
    --------------------------------------------------------------------------------
    1Toshiki Itoh and Pietro de Camilli, “Membrane trafficking: Dual-key strategy,” Nature 429, 141 - 143 (13 May 2004); doi:10.1038/429141a.

    Fish Antifreeze Provided by “Pseudogene” 05/13/2004
    Freezing water forms crystals that can rip and tear at cells. Yet there are fish in arctic waters that can survive even below the freezing point of sea water. They accomplish this by means of special “antifreeze proteins” that interfere with the damaging effects of water crystals.
    Scientists knew about AFP (anti-freeze protein) Type I in winter flounder, and knew its properties. They became puzzled how the fish survived temperatures lower than the protection AFP type I could provide. They suspected another antifreeze protein was at work, and found the gene that codes for it. They explain why this gene, named 5a, had escaped detection for 30 years:

    “The two proteins differ slightly in their amino-terminal sequence and amino-acid composition. At the time of its discovery, the 5a gene was dismissed as an antifreeze-protein pseudogene, largely because the protein it encodes would have been grossly different from type I AFP and had never been detected in the flounder”

    The protein is normally present in low concentrations and degrades at room temperature. At low temperatures, however, it roars into action. It becomes extraordinarily hyperactive, providing more protection against freezing than the previously-known AFP by an order of magnitude.
    --------------------------------------------------------------------------------
    1Marshall, Fletcher, and Davies, “Hyperactive antifreeze protein in a fish,” Nature 429, 153 (13 May 2004); doi:10.1038/429153a.

    Mitochondrial Clock Untrustworthy 05/16/2004
    A major assumption of the “molecular clock” dating method has been called into question. If so, Science Now describes the impact on current theories:

    “ ‘Mitochondrial Eve’, the hypothetical mother of all modern humans who lived about 150,000 years ago, might be lying about her age. A key assumption in determining how long ago she lived—that molecules of mitochondrial DNA do not swap segments with one another—is false, researchers now say. Their findings call into question a multitude of findings in evolution, early human migration, and even the relations between languages.”

    The mitochondria in our cells, organelles that provide the ATP power supply, contain small amounts of DNA. You may have heard that we inherit this mitochondrial DNA only from our mothers. Now, scientists have found evidence that male mitochondrial DNA can be inherited, and might be mixed in with the rest of the mitochondrial DNA. Since “the implications are that this is going on all the time in our cells,” that would render it untrustworthy as a genealogical tracer and dating method.
    An announcement about evidence for recombination in human mitochondrial DNA was published in the May 14 issue of Science.
    --------------------------------------------------------------------------------
    1Kraytsberg et al., “Recombination of Human Mitochondrial DNA,” Science, Vol 304, Issue 5673, 981, 14 May 2004, [DOI: 10.1126/science.1096342].

    Selfish Genes Turn Cooperative 05/19/2004
    Nature1 has reported evidence that transposons help to regulate gene expression. Transposons are genetic material that insert themselves into the DNA of a host, and were thought to represent “selfish genes” that only had their own propagation in mind, “without regard for the consequences.” Some new studies on the L1 retrotransposon, which makes up about 17% of the human genome (mainly within non-coding introns), have shown, however, that they may do us some good.
    The studies “suggest that the insertion of L1 elements into introns can also diminish cellular gene expression in a graded fashion,” the News and Views piece says. “In the words of Han, Szak and Boeke, such L1 insertions provide a ‘molecular rheostat’ with which to govern gene activity — and their bioinformatics analysis establishes that the mechanism is widely used.”
    (For more on the molecular rheostat concept, see 01/10/2003 headline).
    --------------------------------------------------------------------------------
    1Frederic Bushman, “Gene regulation: Selfish elements make a mark,” Nature 429, 253 - 255 (20 May 2004); doi:10.1038/429253b.

    Gene Regulation: When Nonsense Makes Perfect Sense 06/03/2004
    Nature June 31 reports on another use for “junk DNA.” A portion of previously-considered “nonsense” genetic code, which does not produce a protein as does a gene, nevertheless has an important role: it regulates the expression of the neighboring gene. This opens a whole new realm of function for portions of our genetic material that were thought to be useless leftovers of evolution: it’s a new kind of gene that regulates other genes (see the Reuters summary on MSNBC News).
    Molecular biologists have been intrigued by the fact that the DNA translation machinery seems busier than required to produce proteins: “Why is there such a heavy traffic of RNA polymerases, the enzymes that copy DNA into RNA, and the production of large quantities of apparently non-coding and non-functional RNAs?” the reporters say. The new work by Martens et al. reported in the same issue shows that “RNA polymerases are evidently doing more than we thought.” The resulting “nonsense” RNAs produced by reading non-coding segments act as regulators, controlling the amount of protein that is expressed by the true genes by a process of “transcriptional interference.” What was considered nonsense, therefore, actually makes perfect sense on a higher level:

    “Taken together, these studies highlight the importance of intergenic transcription in regulating gene activity, even in the relatively densely packed genome of yeast. It seems that RNA polymerases are not only required for the production of particular RNA species, but by travelling along DNA they can also control the occupancy of regulatory sites by transcription factors. Widespread transcription of intergenic sequences has also been described in the human genome. Surprisingly, many of these non-coding transcripts seem to be regulated in a manner that is intimately connected to the transcription of protein-coding genes. So the high proportion of non-coding regions in the genomes of higher organisms is probably not due to the accumulation of nonsense DNA, but rather represents the evolution of ever more complicated gene-regulatory systems.”

    EurekAlert puts this finding into perspective:

    “If so, the findings would carry an important message for the post-human genome era-namely, that researchers’ attempts to turn the masses of data churned out by the Human Genome Project into an understanding of what actually happens in the human body may be even more complex than they anticipated. One of the main challenges for that effort is to figure out how and when genes are turned on and off during normal development and disease. Rather than look only at how genes are regulated by proteins, they would have to turn their attention to a new, and possibly more-difficult-to-detect form of control. And given that junk DNA makes up 95 percent of chromosomes, the mechanism could be fairly common.”

    The article gives the bottom line to one of the researchers, Fred Winston of Harvard Medical School: “Every time we thought we understood everything going on here, we have been wrong. There are additional layers of complexity.”
    --------------------------------------------------------------------------------
    1Sabine Schmitt and Renato Paro, “Gene regulation: A reason for reading nonsense,” Nature 429, 510 - 511 (03 June 2004); doi:10.1038/429510a.

    DNA Folds With Molecular Velcro 06/07/2004
    Many have heard how the inventor of Velcro got the idea from plant seeds that stick to clothing, but now Carlos Bustamente and team of Howard Hughes Medical Institute have found a velcro-like principle operating at a scale millions of times smaller. Small proteins called condensins are involved in the elaborate folding that DNA undergoes as it is wrapped into chromosomes. The team developed an ingenious method of gently pulling on DNA strands compacted with condensin. Bustamente relates, “when we began to pull it apart carefully, we saw it extend in a sawtooth pattern of force, like the click-click-click of Velcro unzipping.” When they relaxed the force, it collapsed back, then repeated the same pattern when pulled apart again. “That perfect reproducibility strongly suggested to Bustamante and his colleagues that they were seeing a condensed structure with a well defined organization,” the press release explains. Surprisingly, this reversible reaction did not require the expenditure of ATP.

    This is just one of the clever design features in the cell that allows over six feet of fragile DNA to be folded and compacted into a nucleus a few millionths of an inch wide. … Even more amazing is that this tight packing still allows the translation machinery to find the right gene, gain access, and do its work. For a glimpse of this additional complexity, see the 06/13/2002 and 03/08/2002 headlines.

    How Molecular Trucks Build Your Sensors 06/14/2004
    In the film Unlocking the Mystery of Life, biochemist Michael Behe, describing the intricacies of cells as we know them today, claimed that there are “little molecular trucks that carry supplies from one end of the cell to the other.” If that seems an overstatement, you should look at the illustration in Cell June 11 in a Mini-review called “Cilia and flagella revealed” by Snell, Pan and Wang.1 They not only describe trucks, they’ve found a train of boxcars and a whole crew of engineers, conductors and brakemen.
    Cilia are appendages in the cell membrane that wiggle. Everybody’s got them; they are ubiquitous in organisms, from bacteria to humans. They line our respiratory tract, cleaning debris from our lungs. They help our senses of smell and eyesight. They are important for kidney function. They may look simple, but only recently are scientists beginning to appreciate the complexity inside. The authors begin:

    “Our view of cilia has changed dramatically in the decade since Joel Rosenbaum and his colleagues discovered particles rapidly moving (2-4 micrometers/s) up and down within the flagella of the biflagellated green alga, Chlamydomonas (Kozminski et al., 1993). Once cell biologists identified the cellular machinery responsible for this intraflagellar transport (IFT), it became clear that IFT is essential for the assembly and maintenance of cilia and flagella in all eukaryotes (Rosenbaum and Witman, 2002). As we will outline in this brief review, the increased focus on these organelles has revealed that nearly all mammalian cells form a cilium, that the ciliary apparatus (a cilium plus its basal body) is somehow connected with cell proliferation, and that cilia play key (and as yet poorly understood) roles in development and homeostasis.”

    Michael Behe in his book Darwin’s Black Box had a whole chapter on how cilia move. Recently, however, it has been appreciated that nonmotile cilia can also act as sensory probes. The authors explain:

    “Several properties of cilia recommend them for use as sensory transducers. They project a cell type-specific distance from the cell body, making them exquisitely designed probes of the external milieu; both their overlying membrane and their cytoplasmic contents are relatively well isolated from the cell body, thereby offering all of the advantages of compartmentalization; the machinery for their assembly makes possible rapid, regulated transport of proteins between the organelles and the cell body; and, the assembly machinery seems exploitable for use directly in signaling pathways.”

    Now that we know cilia are vital, it’s what goes on inside the narrow shafts during construction that is truly remarkable. The authors mentioned IFT, or intraflagellar transport, a class of proteins that operate the transportation system. During construction of a cilium or flagellum, parts need to be transported to the growing tip, or axoneme. The IFT particles move up and down the inside walls of the shaft. They describe how this works. Watch for the word trucks:

    “This flow of materials is driven by the IFT machinery. Flagellar proteins synthesized in the cell body are carried to the tip of the flagellum (the site of assembly of the axoneme) by IFT particles, which are composed of at least 17 highly conserved proteins that form A and B complexes. The plus end-directed microtubule motor protein kinesin II is essential for movement of particles and their cargo toward the tip (anterograde transport) of the flagellum, and a cytoplasmic dynein carries IFT particles back to the cell body (retrograde transport). Thus, IFT particles function as constantly moving molecular trucks on a closed loop. The tracks they travel on are the microtubule doublets of the ciliary/flagellar axoneme, microtubule motors power them, and the individual structural components (e.g., microtubule subunits, dynein arms, and radial spoke proteins) of the cilium/flagellum are their cargo.”

    The construction system they describe next is reminiscent of a gondola at a ski resort, a series of ore carts in a mine shaft, or a conveyor at a rock quarry. If you can picture architects building a tall structure like the Seattle Space Needle or the Eiffel Tower, imagine the engineers first devising a way to get the raw materials to the growing top. Suppose they design a double trackway that can be extended in length as the structure grows. Attached to this track are self-propelled dump trucks that can climb up the tracks, and another set of dump trucks that can climb down. Each truck can carry a load of cargo. New trucks are constantly added at the bottom, and old ones upon reaching the base are removed. A pool of trucks and drivers is always available to traverse this vertical highway. With this automated system running, workers at the top can take the cargo and build with it, and send waste products down the other side. This two-way transportation system works not only to build the tower, but to dismantle it.

    “Figure 2 presents a model for regulation of assembly, disassembly and for regulation of flagellar length. In this model, the rate of particle entry and the number of particles per unit length are independent of length, and cargo loading is regulated. Thus, in a rapidly growing flagellum (in the extreme case), every particle entering carries cargo, and every particle returning to the cell body is empty. Once the proper length is attained, length control mechanisms engage. At this steady-state length, the number of IFT particles entering and leaving per unit time is unchanged, but the proportion of cargo-loaded IFT particles that enters the flagella comes to equal the proportion of cargo-loaded IFT particles that leaves the flagellum. In a disassembling flagellum, the situation is reversed from that of a growing flagellum, and (in the extreme case) every particle that enters the flagellum is empty and every particle that leaves the tip is full. Thus, by regulating cargo binding to particles at both the base and the tip, and by controlling of assembly and disassembly of axonemal components at the tip (presumably driven by mass action and regulatory proteins), cells specify assembly/growth, steady-state length, or disassembly/resorption.”

    The diagram in their figure shows what look like little ore-carts climbing up to the tip and back. The authors describe next how these tall structures function not only as oars and outboard motors, but as chemical antennae. Experiments have “called to the attention of cell biologists the under-appreciated but hardly insignificant role of cilia in sensory transduction.” Here are some of your body parts that depend on these miniature probes that extend out from the cell into the surrounding environment, sensing what’s out there:

    “Humans experience the environment through cilia in major sensory organs. The outer segments of retinal rod cells are modified, nonmotile cilia, replete with photoreceptors for interacting with light; and the odorant receptors in the olfactory epithelium are peppered over the surface of the cilia of olfactory neurons. Moreover, almost every mammalian cell contains a solitary cilium, called a primary cilium, whose most likely function is in signaling (Pazour and Witman, 2003). For example, many of the neurons in brain contain primary cilia, some of which express receptors for somatostatin and serotonin (Pazour and Witman, 2003). Perhaps the most striking example of the importance of primary cilia in homeostasis [i.e., dynamic equilibrium] comes from work on the epithelial cells of the collecting tubules in the kidney. The primary cilium on each renal tubule cell functions as a flow sensor both in vivo and in MDCK cells in vitro. Bending the cilium causes a large, transient increase in intracellular calcium concentration and a consequent alteration in potassium conductance (references in Boletta and Germino [2003]).”

    Each of these cilia, and many more, are constructed by this molecular transportation system. How many parts are involved in building a cilium? If this system were magnified a hundred million times, children might find this the ultimate Lego toy:

    “New proteomic and genomic studies may finally provide a platform for discovery of most of the as yet unidentified genes that encode ciliary/flagellar proteins. A proteomic analysis of the axoneme of human cilia identified over 200 potentially axonemal proteins (Ostrowski et al., 2002). Several of the proteins were previously identified as being in the axoneme, but many have no homologs or are of unknown function.”

    … From genomic studies, they estimate it would require at least 362 genes to build a motionless cilium, and “more than 400-500 genes that are predicted to be needed for forming and regulating the ciliary apparatus” One team measured the proteome (set of proteins) required to build the basal body (the bottom foundation of the structure) and flagellum to consist of 688 genes. “There is no doubt,” they say, “that the FABB [flagellar and basal body] proteome represents an incredibly rich resource.”
    Failure of cilia and flagella to develop properly are implicated in many diseases (see “Don’t mutate this gene, or else” in the 10/01/2003 headline). Even some human obesity disorders might be traced to ciliary breakdown, as well as hypertension, diabetes and other “seemingly unrelated clinical problems”.
    The authors do not speculate on how such a complex system with so many parts might have evolved, other than to assume that it did: for instance, “Paralogs of other mitotic proteins have also evolved [sic] to play roles [sic] in cilia.” They also claim that plants unevolved them: they seem to have lost the 400-500 genes needed for building cilia or flagella, if they ever had them. The authors examine studies in comparative genomics to determine how many of the cilia/flagella genes are ancestral, going back to the original machinery in the simplest alga or bacterium. One study compared the IFT genes in several organisms with those in fruit flies:

    “Using a large number of genomes provided stringent criteria and identified 187 candidate ancestral ciliary genes. Sixteen are conserved in all ciliated organisms examined and absent in all nonciliated organisms; 18 are present only in organisms with motile cilia; 103 are common to organisms that utilize only conventional ciliogenesis; and 50 are shared only by organisms that form motile cilia in the ciliary compartment.”

    Other studies are cited; 67% of the basal body genes in green algae and 90% of their flagellar and IFT genes were present in the full FABB proteome. It appears, therefore, that this transportation system evolved early on, if it did, and has not changed much since.
    --------------------------------------------------------------------------------
    1William J. Snell, Junmin Pan, and Qian Wang, “Minireview: Cilia and Flagella Revealed: From Flagellar Assembly in Chlamydomonas to Human Obesity Disorders,” Cell, Vol 117, 693-697, 11 June 2004.

    Biochemists Mutate Protein, Make a Catalyst 06/25/2004
    “Enzymes are among the most proficient catalysts known,” wrote three Duke University scientists, “and they catalyze a wide variety of reactions in aqueous solutions under ambient conditions with exquisite selectivity and stereospecificity.” The team set out to rationally design their own enzyme. Their work is reported in the June 25 issue of Science.1 Building on a non-enzymatic ribose-binding protein, they introduced 18 to 22 mutations at specific points, imitating the active site of triose phosphate isomerase (TIM). They succeeded in getting a million-fold increase in catalytic activity, and showed their NovoTim invention to be biologically active in E. coli bacteria. To them, not only does this demonstrate scientists’ ability to understand and imitate “naturally evolved” enzymes, but the “introduction of TIM activity into RBP is therefore equivalent to convergent evolution by computational design.” Their enzyme was less thermally stable than the wild type, however, and the reaction rate was 220 times lower.
    --------------------------------------------------------------------------------
    1Dwyer, Looger and Hellinga, “Computational Design of a Biologically Active Enzyme,” Science, Vol 304, Issue 5679, 1967-1971, 25 June 2004, [DOI: 10.1126/science.1098432].
    Last edited by bob b; October 26, 2006, 06:50 AM.

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    How Do Plants Know When to Bloom? 01/07/2004
    Scientists like to use big words to impress the rest of us, so they have a term for how a plant decides when to bloom: vernalization. But making up a word for a phenomenon is not the same as explaining it.
    Everybody observes that plants seem to just “know” that spring is here, when they put forth their glorious blossoming colors, but think about it: how can a plant, without eyes or a brain or a calendar, judge when it is safe to send out flowers? Through all the vagaries of weather they have an uncanny sense of timing. It’s especially puzzling how winter annuals do this, and biennials, which only bloom in the second year. How can a plant have a memory, and sense the seasons? What goes on in the genes, at the molecular level? How can the memory be preserved through multiple cell divisions?
    This was the subject of two scientific papers in the the Jan. 8 issue of Nature,1,2 and an analysis by Christopher Surridge.3 The process is very complex and still mysterious in many respects. It involves quite a few genes and proteins, particularly histones which are part of the chromatin that wraps DNA, and additional signaling molecules like acetyl and methyl groups. Biochemists have found that, in many cellular processes, there are starters and stoppers: genes and proteins that initiate or suppress an action, and other genes and proteins that stop or re-enable them. For instance, a molecule might clamp onto a gene, making it impossible for the translation machinery to read it, and another molecule will remove the suppressor, allowing the gene to be read and transcribed into a protein. The complex dance of activators and repressors and signalling molecules can be triggered by the external environment and by other activities inside the cell.
    If you can keep this all straight, vernalization goes something like this: a gene named FLC prevents flowering, and is normally expressed during the off-season. A cold snap induces the VIN3 protein to remove acetyl groups from the histones on the chromatin near this gene, signalling two other molecules (vernalization proteins VRN1 and VRN2) that this gene is silenced. Their job is to keep it that way, so that suppression of flowering is itself suppressed. The FLD gene, which promotes flowering, is then expressed. Somehow, FLD tells the molecules at the apical meristem (see 11/20/2003 headline), to send out the buds. Surridge explains, “Silencing is an effective means of controlling long-term gene expression, as it persists even after cells divide. In animals, switching silencing on or off is a well-known way to control development. It seems that plants share this system, using it to preserve the memory of winter’s passing.”
    How does cold cause these reactions? What is known so far is just part of a more involved process. One of the papers2 admits, “How cold results in low FLC RNA and whether any post-transcriptional regulation occurs that feeds back to cause reduced transcription is unknown at present.” The other paper1 says, “The additional components that interact with VIN3, and VRN1 and VRN2, to repress FLC during and after vernalization are not known.” Undoubtedly there are other environmental cues that affect vernalization, such as length of daylight and nutrient availability.
    A popular-level account from Reuters on these results can be found on MSNBC.com.
    --------------------------------------------------------------------------------
    1Sibum Sung and Richard M. Amasino, “Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3,” Nature 427, 159 - 164 (08 January 2004); doi:10.1038/nature02195.
    2Ruth Bastow et al., “Vernalization requires epigenetic silencing of FLC by histone methylation,” Nature 427, 164 - 167 (08 January 2004); doi:10.1038/nature02269.
    3Christopher Surridge, “Plant development: The flowers that bloom in the spring,” Nature 427, 112 (08 January 2004); doi:10.1038/427112a.

    How Enzymes Work – But Don’t Ask Where They Came From: Just Believe 01/12/2004
    Enzymes are protein machines in the cell that speed up reactions that normally would proceed very slowly or not at all. Four biochemists publishing in the Jan. 9 issue of Science1 describe the exquisite power of these biological catalysts: “Enzyme catalysis, which can produce rate accelerations as large as a factor of 10exp19, involves molecular recognition at the highest level of development.” That figure represents a speed increase of 10 quintillion (see also 05/06/2003 headline). After a brief review of efforts to understand enzymes, they remark that “An overview of our present understanding of enzyme catalysis is particularly timely because of the increasing number of articles that propose a variety of origins for enzyme catalysis,” of which they list the names of some proposals. Their paper offers a framework that incorporates these proposals.
    They used rate theory and computer simulations to characterize some of the methods enzymes use to perform their specific reactions. Their table lists sixteen different mechanisms used by sample enzymes from plants and animals. Here is an example for tyrosine-tRNA synthetase:
    Enzyme-transition state and enzyme-intermediate complementarity help to stabilize the transition state of tyrosine activation and to shift the chemical equilibrium by seven orders of magnitude in the direction of the intermediate. Loop motions induced by the chemical process are essential in creating these interactions and permitting access to the active site. (Emphasis added; for more on the tRNA-synthetase family of enzymes, see 07/21/2003 and 06/09/2003 headlines.)
    After providing detailed mathematical analyses of these mechanisms, they conclude, “Evolutionary selection makes possible the development of enzymes that use a wide range of molecular mechanisms to facilitate reactions. Although, in principle, such rate enhancements could arise from lowering the quasithermodynamic free energy of activation or increasing the generalized transmission coefficient, the present analysis shows that the former plays the dominant role” (emphasis added). They feel that modern transition state theory is adequate to describe these processes.
    --------------------------------------------------------------------------------
    1Garcia-Viloca, Gao, Karplus, and Truhlar, “How Enzymes Work: Analysis by Modern Rate Theory and Computer Simulations,” Science 09 Jan 2004, 10.1126/science.1088172.

    Centromere Shows More Gems in “Junk DNA” 01/12/2004
    A biochemist at University of Wisconsin-Madison and a colleague sequenced a hard-to-sequence part of the rice genome, the centromere, and found four genes in it. Previously, it was thought to be a vast wasteland of repetitive, non-coding DNA. The scientist, Jiming Jiang, thinks his work provides a “window to evolution” of the centromere, according to writer Terry Devitt: “The evolutionary progression [sic] of the centromeres, Jiang suggests, may be analogous to how temperate forests evolve [sic] from more diverse ecosystems to climax forests where a single species of tree dominates. In the rice centromere, it may be that evolution has not yet purged active genes to be replaced by the long and repetitive blocks of DNA that mark the centromeres of most organisms”.

    Live at the Improv: DNA Polymerase 01/20/2004
    When a DNA reader hits an unfamiliar line, it improvises, reports EurekAlert:
    Prof. Zvi Livneh and Ph.D. student Ayelet Maor-Shoshani of the Biological Chemistry Department cut a DNA strand — from the bacterium E. coli — and inserted material similar to that which composes crude oil in between both its ends. As expected, the regular DNA polymerase stopped working when it reached the foreign material. Yet to the scientists’ amazement, a specialized DNA polymerase jumped in to rescue the stalled replication process, and continued the copying process, inserting nonexistent genetic components into the “printout’ when it encountered the foreign material. This can be compared to a person who forgets some words in a song and makes up new ones to be able to continue to sing.
    The scientists believe this capability provides resilience against damaged DNA, except in the most extreme cases:
    True, when DNA polymerase improvises a tune, errors (i.e. mutations) may occur in the new cells’ DNA. Yet Livneh explains that the body cannot feasibly let all cells with damaged DNA die, for there are too many of them. “Only if the DNA contains a very high level of damage will the cell’s machinery ‘give up’ and let the cell die.”

    “Utmost Precision” Found in DNA Repair Enzyme 02/13/2004
    The cell has many helper enzymes that can repair DNA damage. One such enzyme, named MutY, has been described in the Feb. 12 issue of Nature.1 Reviewer Tomas Lindahl sets the stage: “Damaged DNA must be removed with the utmost precision, as mistakes are costly. The structure of a repair enzyme bound to its substrate provides a welcome clue to how this is achieved.”
    This particular enzyme is able to recognize its particular error target, an adenine incorrectly paired to an oxidized guanine, because of “extensive and precise contacts” it makes with that specific erroneous pair. These contacts prevent it from mistakenly removing a correct pair. In a paper in the same issue, Fromme et al.2 describe “an ingenious way by which this specificity is achieved” through these multiple, precise contacts.
    Lindahl describes how this enzyme works. Details of the jargon are not essential for appreciating the precision of this molecular machine’s lifesaving activity:

    “MutY belongs to a group of enzymes known as DNA glycosylases, which recognize altered bases in DNA and help to remove them. Like other DNA glycosylases, it generates a sharp bend in the DNA at the site of the mismatch. The new structural data provide a suitable explanation for why — as is desired — MutY doesn’t recognize and remove an adenine opposite its normal base partner, thymine (T): the extensive and precise contacts between MutY and an A•xoG pair are entirely absent in a normal AT pair. Similarly, the enzyme’s active site does not accommodate a cytosine opposite an oxoG; for coding reasons, it is important that the oxidized base rather than the normal base is repaired in this partnership.”

    Lindahl notes that mutations in this enzyme put humans at risk of colorectal cancer. Other oxygen-altered bases, if not repaired, are implicated in tissue degeneration and ageing.
    --------------------------------------------------------------------------------
    1Tomas Lindahl, “Molecular Biology: Ensuring error-free DNA repair,” Nature 427, 598 (12 February 2004); doi:10.1038/427598a.
    2Fromme et al., Nature Feb 12, 2004, p. 652.

    Your Internal Motors Can Run Nanotech 02/13/2004
    In each cell in your body, and in that of every living thing, there exists a tiny motor named ATP synthase that Science News1 calls “the ultimate molecular machine.” It converts electrical to chemical energy, writes Alexandra Goho, “with amazing efficiency.” Now, Japanese have harnessed some of these motors (only 12 millionths of a millimeter high) to power artificial machines. They attached hundreds of the motors to a glass surface and attached little magnetic beads to the rotor part. With an electromagnet, they induced them to spin, and were able to make them rotate both clockwise and counterclockwise.
    --------------------------------------------------------------------------------
    1Alexandra Goho, “Nature’s tiniest rotor runs like clockwork,” Science News, Week of Feb. 7, 2004; Vol. 165, No. 6, p. 94; see also article by Jessica Gorman, “Nanotech Switch: Strategy controls minuscule motor,” Science News, Week of Nov. 9, 2002; Vol. 162, No. 19.

    DNA Is a Code Operated by Another Code 02/17/2004
    The discovery in the 1950s that DNA stored a coded language was amazing, but recently a new level of complexity has come to the awareness of biochemists. Apparently, another code determines which DNA genes will be opened for expression and which should be suppressed.
    The Feb. 14 issue of Science News1 describes the history of the discovery of the so-called “histone code.” These are patterns of “tails” attached to the histones around which DNA is tightly wrapped. Within the last eight years, scientists have been discovering that the histones do not merely spool the DNA, they regulate which genes get expressed.
    The pattern of acetylation and methylation on the histone tails appears to form a code that is heritable through cell divisions. Compared to the well-known DNA genetic code, “A histone code may be much more complex,” writes John Travis. Shelley Berger (Wistar Institute) exclaimed, “There are all kinds of sites [on histone tails] that can be modified. The possibilities for a code are quite enormous. It’s not going to be a simple code.” After summarizing the literature, Travis concluded, “With such designer histones, it seems that researchers are on their way to having in their hands all the words of the histone code. But, it may still be a stiff challenge to figure out what those words mean.”
    For a previous story on the histone code, see 11/04/2002 headline, “Cell Memory Borders on the Miraculous.”
    --------------------------------------------------------------------------------
    1John Travis, “Code Breakers: Scientists tease out the secrets of proteins that DNA wraps around,” Science News, Vol. 165, No. 7, Feb. 14, 2004, p. 106.

    Cellular Cowboys: How the Cell Rounds Up Chromosomes Before Dividing 03/04/2004
    Two cancer researchers from UC San Diego describe mitosis (cell division) in the Mar. 4 issue of Nature.1 Pulling together the latest findings about this elaborate and important process, they begin by describing the puzzle that the cell needs to solve:

    “At the beginning of mitosis, the process of cell division, chromosomes are organized randomly — like jigsaw puzzle pieces spread out on the floor. Their constituent two ‘sister chromatids’, each of which contains one of the two identical DNA molecules produced by replication, must be oriented such that they will be pulled in opposite directions into the two newly forming cells. Like a jigsaw, the solution for correctly orienting all chromosomes comes partly through trial and error. Mechanisms must exist to eliminate wrong configurations while selecting the right ones.”

    In the article, they describe how cables (microtubules) connect to handles (kinetochores) on the chromosomes and start pulling them in opposite directions. Another enzyme dissolves the molecular “glue” in the centrosomes that hold the sister chromatids together, so that the opposite poles of the spindle can pull them apart into the daughter cells.
    A newly-described “highly-conserved enzyme” (i.e., identical in yeast and vertebrates), named Aurora B kinase, somehow finds chromosomes that lack an attachment to the other pole of the spindle, and fixes them. Apparently this enzyme is able to identify chromosomes that are incorrectly lassoed to the same pole (syntelic attachment) and therefore are not under tension. Only when there is tension on each chromosome, pulling the sister chromatids toward opposite poles, will the process continue. “Finding out how Aurora B identifies and corrects them is an obvious next step,” the authors say.
    --------------------------------------------------------------------------------
    Ian M. Cheeseman and Arshad Desai, “Cell division: Feeling tense enough?”, Nature 428, 32 - 33 (04 March 2004); doi:10.1038/428032b.

    Cell Networks 03/22/2004
    A team of Chinese scientists analyzed protein interactions in yeast cells, and titled their paper in PNAS1 “The yeast cell-cycle network is robustly designed.” They “demonstrated that the cell-cycle network is extremely stable and robust for its function,” and “able to survive perturbations.” The beginning of the paper expresses the wonder the stimulated their research:

    “Despite the complex environment in and outside of the cell, various cellular functions are carried out reliably by the underlying biomolecular networks. How is the stability of a cell state achieved? How can a biological pathway take the cell from one state to another reliably?

    After analyzing the stable states, “big attractors” and checkpoints in the yeast cell cycle, the scientists remind us that this network is part of an even bigger marvel:

    “Note that the network we studied ... is only a skeleton of a larger cell-cycle network with many ‘‘redundant’’ components and interactions.... Thus, we expect the complete network to be even more stable against perturbations.
    ... Furthermore, our results suggest that not only do biological states correspond to big fixed points but the biological pathways are also robust.
    Functional robustness has been found in other biological networks, e.g., in the chemotaxis of E. coli (in the response to external stimuli) and in the gene network setting up the segment polarity in insects development (with respect to parameter changes) . It has also been found at the single molecular level, in the mutational and thermodynamic stability of proteins. In some sense, biological systems have to be robust to function in complex (and very noisy) environments.”

    And now to the climax. In the closing statement, after claiming several times that these networks are “robustly designed” (their term), they suggest that all this complexity, all this robustness, all this control and regulation is the product of time, chance and contingency. In fact, the very robustness might even help evolution make it better:

    “More robust could [sic] also mean more evolvable, and thus more likely to survive [sic]; a robust ‘‘module’’ is easier to be modified, adapted, added-on, and combined with others for new functions and new environments. Indeed, robustness may provide us with a handle to understand the profound driving force of evolution.”

    --------------------------------------------------------------------------------
    1Fangting Li, Tao Long, Ying Lu, Qi Ouyang, and Chao Tang, “The yeast cell-cycle network is robustly designed,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0305937101, published online before print March 22, 2004.

    Quick Picks03/31/2004
    Too many stories came in too fast at the end of March. Here are some we would have liked to explore in more detail. They’re all interesting and some have amazing facts and quotes.

    DNA vs. Evolution: A paper in the Royal Society Biology Proceedings1 warned that pleiotropy, the antagonistic effect of genes that need to mutate together, inhibits natural selection more than is usually realized. Sarah P. Otto writes,

    “Pleiotropy is one of the most commonly observed attributes of genes. Yet the extent and influence of pleiotropy have been underexplored in population genetics models. ... Under the assumption that pleiotropic effects are extensive and deleterious, the fraction of alleles that are beneficial overall is severely limited by pleiotropy and rises nearly linearly with the strength of directional selection on the focal trait. Over a broad class of distribution of pleiotropic effects, the mean selective effect of those alleles that are beneficial overall is halved, on average, by pleiotropy.”

    Thus the simplistic notion that a beneficial mutation will be acted on by natural selection is “severely limited” by the effect of pleiotropy.

    Another Thing You Can’t Live Without: David Carling (Imperial College) provides a quick review of AMPK in the 23 March issue of Current Biology5. If you don’t know what AMPK is (AMP-activated protein kinase), just be glad you (and everything else alive) has it:

    “AMPK has been dubbed the cellular fuel gauge, because it is activated by a drop in the energy status of the cell. If ATP is used up faster than it can be re-synthesized, ATP levels fall and this leads to a rise in AMP. The increase in the AMP:ATP ratio triggers the activation of AMPK and leads to the phosphorylation of a large number of downstream targets. The overall effect of AMPK activation is to switch off energy-using pathways and switch on energy-generating pathways, thus helping to restore the energy balance within the cell. The conservation of AMPK throughout evolution emphasises its importance: homologs have been identified in all eukaryotic species examined to date, including plants.”

    Other recent articles have focused on this cellular “fuel gauge” as a means of controlling appetite and obesity (see, for instance, Nature April 1, 2004). When asked “Can we live without it,” Carlin answers immediately, “Almost certainly not.” Mice without it die in embryo, and it cannot be mutated much: “Although a complete loss of AMPK activity is lethal, subtle changes in AMPK activity can lead to serious clinical consequences.”

    Genome Size: In Current Biology6 Brian Charlesworth and Nick Barton examine the question of why genome sizes differ so much between organisms, and offer a suggestion:

    “Genome sizes vary enormously. This variation in DNA content correlates with effective population size, suggesting that deleterious additions to the genome can accumulate in small populations. On this view, the increased complexity of biological functions associated with large genomes partly reflects evolutionary degeneration.”

    Intron Origins: Another paper in the same issue of Current Biology7 attempts to put forward a hypothesis about intron origin and evolution (see 09/23/2003 headline). “Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns.”
    --------------------------------------------------------------------------------
    1Sarah P. Otto, “Two steps forward, one step back: the pleiotropic effects of favoured alleles,” Proceedings: Biological Sciences, The Royal Society, Issue: Volume 271, Number 1540, April 07, 2004 Pages: 705 - 714 DOI: 10.1098/rspb.2003.2635 (published online before print).
    5David Carling, “:Magazine: AMPK,” Current Biology, Vol 14, R220, 23 March 2004.
    6Brian Charlesworth and Nick Barton, “Genome Size: Does Bigger Mean Worse?” Current Biology, Vol 14, R233-R235, 23 March 2004.
    7Sadusky et al., “Exon Junction Sequences as Cryptic Splice Sites: Implications for Intron Origin,” Current Biology Vol 14, 505-509, 23 March 2004.
    Last edited by bob b; October 25, 2006, 07:15 AM.

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    The Mystery of Intron Evolution 09/03/2003
    Eukaryotes, the organisms whose cells contain a nucleus, encompass an astonishing diversity of organisms: all plants and animals and a number of one-celled protists. The genomes of eukaryotes contain a mystery: sections of DNA, called introns, that do not code for genes, and are exquisitely removed before translation by a complicated molecular machine called the spliceosome (see 09/12/2002 headline) and its helpers. The spliceosome, a large RNA-protein complex, is conserved throughout the eukaryotic world, from grizzly bears to earthworms to orchids. Coding regions of the eukaryotic DNA, called exons, are meticulously spliced together after the introns are removed. Why are these introns there? What is their function, if any? Why does the cell go to so much work to remove them? Evolutionary biologists want to know.
    In the Sept. 2 issue of Current Biology1, a team of British and American scientists delved into published genomes to compare intron counts and positions, in hopes of determining the evolutionary history of introns. To their surprise, they found “Remarkable Interkingdom Conservation of Intron Positions and Massive, Lineage-Specific Intron Loss and Gain in Eukaryotic Evolution,” as the title of their paper summarizes. They compared eight very different eukaryotes: the malaria parasite and a cousin, two kinds of yeast (fungi), Arabidopsis (a flowering plant favored in genetics research), fruit flies, worms, and humans. Sifting through the published genomes, they found 684 orthologous sets but no clear evolutionary pattern. Surprises include:

    • More introns share identical positions within two or more species than would be expected by chance.
    • Intron phylogeny does not match traditional Darwinian phylogeny.
    • Humans share more introns with the herb, a plant, rather than with animals like fruit flies and worms.
    • Yeast and malaria also both share more introns with plants than with fruit flies and worms.
    • The number of introns in different species varies widely. Humans and plants have many, but yeast and fruit flies have fewer.
    • The majority of plant and animal introns appear to have been gained recently. Humans, for instance, have longer introns than mice (see 12/06/2002 headline).
    • Recently, introns were found in several protists (see 02/26/2002 headline), which “might be the deepest branches in eukaryotic phylogeny.”

    Their surprise is evident in their summary of the findings :

    “The matrix of shared introns in all pairs of analyzed eukaryotic genomes revealed a striking, unexpected pattern (Table 2). The number of conserved introns did not drop monotonically with the increase of the evolutionary distance between the compared organisms. On the contrary, human genes shared the greatest number of introns not with any of the three animals but with the plant Arabidopsis; in the conserved regions (the more accurate results given the uncertainties in alignment in other parts of genes), 24% of the analyzed human introns were shared with Arabidopsis (27% of the Arabidopsis introns) compared to 12%–17% of the introns shared by humans with the fly, mosquito, and the worm (Table 2). The difference becomes even more dramatic when the numbers of introns conserved in Arabidopsis and each of the three animal species are compared: approximately three times more plant introns have a counterpart in humans than in the fly or the worm (Table 2). Although S. pombe [yeast] and Plasmodium [malaria] have few introns compared to plants or animals, the same asymmetry was observed for these organisms: the numbers of introns shared with Arabidopsis and humans are close and are 2–3 times greater than the number of introns shared with the insects or the worm (Table 2).”

    How does an evolutionist make sense of these unexpected findings? The team applied phylogenetic tree-building to the data, and quickly realized that intron presence or absence does not fit Darwinian expectations. A method called Dollo parsimony, for instance, assumes that each derived character state (e.g., intron presence) originated only once on the tree. This led to a very unDarwinian tree, with humans clustered together with plants, and yeast clustered with malaria. They tried other phylogenetic approaches that generated more of a web of branches going nowhere than a tree progressing toward higher organisms (see their Figure 2). “These observations show that intron locations are not suitable markers for phylogenetic analysis at long evolutionary distances,” they confessed.
    “Having shown that evolution of introns in eukaryotic genes did not follow the species tree,“ they inverted the analysis and started by assuming the species tree to deduce the evolution of introns. This led to a conclusion that the “last common ancestor [sic] of the eukaryotic species with sequenced genomes comes out particularly intron rich,” they note. In other words, introns and the spliceosome machinery to juggle them were already present in the earliest eukaryote. For some reason, certain groups lost introns, and others had a net gain, while not a few introns survived nearly two billion years of evolution intact and in their original positions.
    Why would some species conserve introns, and others get rid of them? The authors consider possibilities. “Why have so many ancestral [sic] introns survived almost 2 billion years of evolution?” they ask. “One intriguing possibility is that conserved introns are functionally important, but there is currently little evidence in support of this hypothesis.” On the other hand, maybe losing introns, even if functionless, is hazardous; like trying to remove a bullet, maybe it is sometimes better to leave it in than to risk more damage by removing it. Perhaps their presence influences gene regulation and expression (see 07/22/2002 headline). If so, removing them could be lethal. But then, why would some organisms, like fruit flies, succeed in removing so many of them without harm? No clear reason is given why natural selection would favor removing introns on some groups and adding them in others.
    Without a clear phylogeny emerging from the data, they conclude with some speculations. “It even seems possible,” they imagine, “that invasion [sic] of protein-coding genes by ancestors of introns was part of the dramatic and still mysterious series of events that led to the origin of the eukaryotic cell.” The bottom line is that this “remarkable conservation” across species – and even kingdoms – leaves much to be learned: “The lineage-specific trends of intron loss and gain might reflect more general tendencies for genome compaction and genome expansion, the underlying causality of which remains to be understood.”

    Update: see Sept. 12 headline.
    --------------------------------------------------------------------------------
    1Rogozin, Wolf, Sorokin, Mirkin and Koonin, “Remarkable Interkingdom Conservation of Intron Positions and Massive, Lineage-Specific Intron Loss and Gain in Eukaryotic Evolution,” Current Biology Vol 13, 1512-1517, 2 September 2003

    Left-Handed Amino Acids Explained? 09/06/2003
    Another theory has surfaced to explain the origin of left-handed amino acids in proteins. Reported in Science News1, R. Graham Cooks and colleagues at Purdue University studied all 20 biological amino acids, and found that one – serine – formed stable clusters of all left- or all right-handed forms. The third lightest (after glycine and alanine), possessing an uncharged polar side chain, serine not only clustered in single-handed forms, but attracted other amino acids of the same hand. Sugars of the opposite hand were also attracted to the eight-molecule serine rings.

    “Serine clusters’ high stability and selectivity have convinced the researchers that left-handed serine must have forced its chemical siblings to follow its lead [sic]. What caused serine’s left form to become dominant in the first place remains an open question. Some scientists say that ancient minerals may have favored one form over the other (SN: 5/5/01, p. 276). Others point to the effects of radiation hitting primordial Earth. Or, says Cooks, it could have happened by chance.”

    As to this chance event, Cooks speculates in the Purdue News press release that “If somehow polarized light, for example, or a swirling motion in water were present at a critical moment, some of the right-handed clusters could have become left-handed. This could have cascaded into other prebiotic reactions and set the pace for a billion years of evolution” [sic]. He calls serine the “bouncer at life’s dance club.” His team’s paper was published online Aug. 4 in the German chemistry journal Angewandte Chemie.2
    --------------------------------------------------------------------------------
    1Science News Week of Sept. 6, 2003 (164:10): Alexandra Goho, “Amino acid lends a heavy hand.”
    2Takats, Z., S.C. Nanita, and R.G. Cooks, “Serine octamer reactions: Indicators of prebiotic relevance,” Angewandte Chemie International Edition, Volume 42, Issue 30, Pages 3521 - 3523 (published online Aug. 4).

    Cells Fight Mutations 09/12/2003
    A team of cancer researchers identified 33 genes in yeast “potentially associated with the suppression of the accumulation of mutations.” Their paper was published online Sept. 12 in the Proceedings of the National Academy of Sciences1, where they begin by stating,

    “Maintaining the stability of the genome is critical to cell survival and normal cell growth. Inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer as evidenced by the observation that a number of the DNA-repair and checkpoint genes are mutated in cancer susceptibility syndromes and sporadic cancers. This raises the possibility that other genetic defects causing genome instability and mutator phenotypes could contribute to carcinogenesis.”

    Their blind screening technique found all the known mutation suppressors, but also ten more previously-unknown “nonessential” genes involved in mutation suppression.

    “Of the confirmed genes, five encode components of the oxidative-stress response, and six are genes of unknown function. We believe the data presented here define a nearly complete collection of nonessential genes involved in suppression of mutations in the CAN1 forward-mutation assay [a sample gene used for evaluating effects of mutations] and define several previously unappreciated mutation-suppression pathways.”

    Some of the activities these genes perform are repairing base excisions, fixing oxidated guanine (G) bases, and replacing incorrectly-inserted uracil (U) bases with cytosine (C). Another one suppresses genome rearrangements.
    --------------------------------------------------------------------------------
    Huang, Rio, Nicolas and Kolodner, “A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.2035018100.

    Intron Update 09/12/2003
    (See Sept. 3 headline about introns). Scientists have found a possible reason why genes that contain introns are expressed more effectively than those without. Writing in PNAS1, a team from Howard Hughes Medical Institute suspects that the exon junction complex (EJC) that forms at each junction by the splicing process may give messenger RNA (mRNA) a tethering point with “position-specific memory of the splicing event.” EJC components may attach at the junctions to perform expediting functions.
    Of five known protein components of the EJC, some are known to be involved in nonsense-mediated decay, positioning of the mRNA in the cytoplasm, or transport through the nuclear pore complex (see March 4 headline). Other functions of the EJC might include stabilizing the mRNA, enhancing transcription in the ribosome, and translational utilization.
    They found that gene expression was enhanced by the EJC components but not necessarily by the act of splicing itself; that is apparently why some intronless genes can be expressed satisfactorily, especially if EJC proteins can be recruited through other means. Splicing may assist in the efficient formation and localization of EJC components. The stimulatory effect of splicing and the EJC can enhance gene expression more than 30-fold.
    --------------------------------------------------------------------------------
    1Wiegand, Lu, and Cullen, “Exon junction complexes mediate the enhancing effect of splicing on mRNA expression,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.1934877100.

    Plasma Blob: It’s Alive? 09/17/2003
    New Scientist claims, “Plasma blobs hint at new form of life.” In an experiment worthy of Frankenstein, a Romanian scientist inserted two high-voltage electrodes into a plasma of argon, and created cell-like spheres of plasma that could replicate by splitting in two and “communicate information” by emitting electromagnetic energy, making atoms in other spheres vibrate.
    Mircea Sanduloviciu and team (Cusa University, Romania) claim this shows that “cell-like self-organization can occur in a few microseconds” instead of millions of years. Sanduloviciu speculates these could have been the first cells on earth, formed in electrical storms. “The emergence of such spheres seems likely to be a prerequisite for biochemical evolution,” he said. Others think that is a stretch, but are intrigued by the implications that life might take on forms much different than the DNA and protein-based life we know on earth.

    Your Motors Perform Cooperative Interactions 09/18/2003
    The motor that powers all life, often called a “splendid molecular machine,” looks even more splendid due to research by Caltech and French scientists. It has parts that help each other out. ATP synthase, which we have reported on frequently before (see 11/15/2002 headline), is a true rotary motor, and probably the most abundant enzyme on earth. Its job is to generate ATP, the energy currency of life. “Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology,” they state in their PNAS paper.1 They found that the structure of the six-lobed F1 upper unit, where ATP is catalyzed from ADP and phosphate, actually is tuned to enhance the productivity of the system.
    Most living things have the F0-F1-ATPase model, composed of an upper and lower mechanism joined by a camshaft and some other parts, although there are variations in some bacteria. The six lobes of the F1 motor, arranged in pairs like orange slices around the camshaft, form three catalytic sites where ATP is synthesized. The researchers did thermodynamic and kinetic modeling of these structures and found that the shapes of the sites change as the camshaft rotates in a way that enhances productivity. Each pair of lobes cycles through three stages as the shaft turns: (1) insertion of ADP + P, (2) catalysis of ATP, and (3) ejection of the product. Each stage is not only finely tuned for its job, but actually stimulates the adjoining pair of lobes to do its job better: e.g., at stage one, the shape of the lobes causes the reaction in stage two of the adjoining lobes to accelerate. The reaction in stage two speeds up the ejection of product in stage three, and so on. Overall, this enhances the productivity of the system by a factor of 300 or more than would occur if a pair of lobes had to work alone. This and the rotation of the camshaft enhances productivity by a factor of 500,000.
    They mention some other interesting facts about ATP synthase in passing. Each motor (and your body has quadrillions of them) can hydrolyze from 40 to 600 ATP per second. With three ATP per revolution, that translates to 12,000 RPM at top speed. Without these enzymes, it would take 500,000,000 times as long for ATP to hydrolyze in solution. Though they studied primarily the hydrolysis cycle, the synthesis reaction, driven by an electric current (proton flow) in the lower F0 subunit, is similarly accelerated because of the efficient mechanical arrangement of the parts.
    --------------------------------------------------------------------------------
    1Gao, Yang, Marcus and Karplus, “A model for the cooperative free energy transduction and kinetics of ATP hydrolysis by F1-ATPase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.1334188100, published online 09/18/2003.

    Don’t Mutate This Gene, Or Else 10/01/2003
    Hit the power grid, and you shut down hospitals, businesses and homes. When traffic controllers go on strike, trucks pile up on the highways, and merchants, from florists to toolmakers, cannot get their goods. A whole city or economy can grind to a halt from one failure in a key component. Similarly, mutations to certain genes can have what are called pleiotropic effects, causing harm to very different organs and functions scattered all over the body. In the Sept. 30 issue of Current Biology1, three New York geneticists describe the failures that ensue from a mutation in one protein in the IFT family: “kidney cysts, photoreceptor degeneration, skeletal abnormalities, and spermatogenesis defects. ... A targeted Tg737 knockout [to a particular IFT protein] is embryonic lethal, with early embryonic defects that include randomized left/right asymmetry, a consequence of missing cilia on the embryonic ventral node.” IFT proteins might also be essential for coordinating signals from cilia to other parts of the cell.
    Like most scientific papers, this one is focused on just one narrow aspect of one protein in one organism, the fruit fly. As background information, however, they describe what IFT does and how it works. IFT stands for intraflagellar transport. It is a family of proteins involved in building the insides of flagella and cilia. (These are the whip-like appendages common in most living things, from the outboard motors on bacteria, to the sweepers lining your respiratory tract, to the paddling tails on sperm cells). If you could shrink yourself down to a few microns and watch, here’s what you might see going on inside the shaft of a cilium or flagellum under construction :

    “The eukaryotic cilium or flagellum is a distinct subcellular compartment, with its own characteristic microtubular cytoskeleton, the axoneme, and a membrane that, though continuous with the plasma membrane, can localize distinct sets of proteins. This distinction is maintained by a specific mechanism of intraflagellar transport (IFT). IFT was first observed in the single-celled alga Chlamydomonas as a bidirectional movement of uniformly sized particles along the flagellum, in the space between the axoneme and the flagellar membrane. Biochemical characterization of the particles revealed over 16 constituent proteins associated in A and B subcomplexes. Particle movement toward the plus ends of the axonemal microtubules at the tip of the flagellum is driven by kinesin II, and mutants lacking kinesin II subunits or complex B proteins do not extend cilia beyond the transition zone of the basal body. In mutants that express a temperature-sensitive kinesin, flagella shrink after a shift to the restrictive temperature, and this shrinkage indicates that IFT is needed to maintain and regulate flagellar length. IFT particles and kinesin are returned to the cell body by a nonaxonemal dynein, and mutants with defects in this process typically have swollen cilia that accumulate IFT particles. Some IFT proteins are concentrated in the cytoplasm close to the basal bodies as well as in the cilia proper, and the transition fibers that connect the basal body to the cell membrane are a possible site for the docking and exchange of IFT particles, motors, and cargo.”

    In other words, there is a specialized molecular highway down the shaft of a flagellum, between the membranes, with little molecular trucks (dynein and kinesin) that transport cargo (the protein particles) to and from the tips of the growing end. Intraflagellar transport might be termed the Transportation Department for these organelles. Since everything from eyes, sperm, and lungs depend on cilia or flagella, you can imagine what happens when a mutation shuts down the highway department and brings construction of these essential organelles to a halt.
    A related paper in the same issue2 discusses what happens when another one of the IFT proteins, Kinesin II-mediated anterograde intraflagellar transport, mutates and prevents the kinesin truck from moving down the highway. It makes their cilia sluggish and uncoordinated, and causes auditory defects.
    An analysis by George Witman (U. of Mass. Medical School) of these papers was published in the subsequent (Oct. 14) issue of Current Biology.3
    --------------------------------------------------------------------------------
    1Han, Kwok and Kernan, “Intraflagellar Transport Is Required in Drosophila to Differentiate Sensory Cilia but Not Sperm,” Current Biology Vol 13, 1679-1686, 30 September 2003, pp. 1679-1686.
    2Sarpal et al., “Drosophila KAP Interacts with the Kinesin II Motor Subunit KLP64D to Assemble Chordotonal Sensory Cilia, but Not Sperm Tails,” Current Biology Vol 13, 1687-1696, 30 September 2003, pp. 1679-1686.
    3George B. Witman, “Cell Motility: Deaf Drosophila keep the beat,” Current Biology Vol 13, R796-R798, 14 October 2003.

    Mitochondrial Ribosome Structure Casts Doubts on Endosymbiont Theory 10/07/2003
    Have you heard the story that early cells swallowed other ones and made them their slaves? That is supposedly where mitochondria came from, but an article in the Oct. 3 issue of Cell reports that there are some big differences between the mitochondrial ribosomes of eukaryotes and those of bacteria, the presumed captives.
    Manjuli Sharma et al.1 determined the structure of the eukaryotic mitochondrial ribosome (mitoribosome) for the first time. These ribosomes (sites of protein synthesis) differ from those in the cytosol, because they produce 13 specialized proteins dedicated primarily to the production of ATP.

    “According to several genomic analyses, mitochondria are believed to have arisen from an early endosymbiotic event between a eubacterium and its host cell .... Therefore, it has generally been expected that the mitoribosome will display greater structural and functional similarities to a bacterial ribosome than to a eukaryotic cytoplasmic ribosome.”

    They found, “However, the RNA and protein composition of the mitoribosome differs significantly from that of bacterial ribosomes.” Whereas the small subunit has 950 nucleotides and 29 proteins, the bacterial counterpart has 1542 and 21, respectively. The large subunit has 1560 nucleotides and 48 proteins, but the bacterial counterpart has 120 + 2904 nucleotides in two units, and 33 proteins. “Thus, the protein-to-RNA ratio is completely reversed in the mitoribosome (69% protein and 31% RNA) relative to bacterial ribosomes (33% protein and 67% RNA),” they note. Even among the roughly half of the proteins in the eukaryotic mitoribosome that have homologs in bacteria, they are usually significantly larger. And the whole ribosome, though larger, is more porous than the bacterial one.
    The rest of the paper describes the functional units of the mitoribosome. They found exquisite entrance tunnels for the transfer RNA and messenger RNA, and precision exit tunnels for the nascent polypeptides. They feel their analysis “provides new insights into the structural and functional evolution of the mitoribosome.” But the paper also describes large differences between the mitoribosomes and the ribosomes in the rest of the cell:

    “Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.”

    It appears, therefore, that these three classes of ribosomes are quite different from each other. This is probably due to the different jobs they have to do, as expressed in the title of their paper: the component proteins of the mitoribosome suggest they have “an expanded functional role” over their counterparts.
    --------------------------------------------------------------------------------
    1Sharma et al., “Structure of the Mammalian Mitochondrial Ribosome Reveals an Expanded Functional Role for Its Component Proteins,” Cell Vol 115, 97-108, 3 October 2003.

    A Self-Regulating Recycling System Found in the Cell 10/07/2003
    Cells are not watertight sacks; they import and export things. But they are not leaky sacks either: everything coming and going is authenticated by sophisticated mechanisms. Small packages, like water molecules or individual proteins, have specially-designed channels embedded in the cell membrane that check their credentials and make them run an electronic gauntlet (see 03/12/02 headline, for instance).2 Larger packages, however, have a surprising method of making their entrance: they dive in and get wrapped in geodesic spheres. The cell membrane neatly reseals itself around the point of entry, which occurs only where specialized receptors allow it. This is called endocytosis (for cargo on the way in) and exocytosis (on the way out).
    The geodesic spheres are made up of a three-armed protein called clathrin. The clathrin molecules envelop the cargo, forming a crystalline polyhedron around it. (You absolutely have to see this cool animation by Allison Bruce of Harvard, showing clathrin forming a spherical vesicle; incredible.) Once the cargo in its crystalline cage has been safely ferried to its destination, the clathrin molecules disassemble and are available for re-use. (This process, and much more, is beautifully illustrated in the award-winning animated short film Voyage Inside the Cell). Exocytosis is the process in reverse, when the cell needs to export cargo to the outside: for example, when a nerve cell needs to send neurotransmitters to another neuron. A host of helper enzymes are involved in making both processes work.
    “Clathrin-mediated endocytosis is one of the primary mechanisms by which eukaryotic cells internalize nutrients, antigens, and growth factors and recycle receptors and vesicles,” begin a team of Pennsylvania scientists in a paper in the Oct. 3 issue of Cell.1 But it should be obvious that the amount of cargo coming in must balance that going out, or else the cell will burst or shrivel. “A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function,” they write, speaking especially of neurons that execute these processes continuously in the central nervous system and the brain. How can the cell maintain this balance?
    These scientists discovered an automatic regulatory process that ensures the materials are recycled properly. A protein called endophilin, a key regulator of the endocytosis process, has two states: open and closed. In the open state, it attaches to the interior side of voltage-gated calcium channels (these are membrane turnstiles that allow only doubly-ionized calcium to pass through). Here, it somehow recruits other protein machines needed for the endocytosis operation. When the calcium concentration reaches 1 micromolar, the endophilin switches into the closed position. Then, it detaches from the calcium gate, “which would presumably allow the liberated endophilin and dynamin [another helper enzyme] to become actively involved in endocytosis immediately after SV [synaptic vesicle] exocytosis.” A similar self-regulating system had been known for exocytosis, but this is the first time a mechanism has been found to regulate endocytosis: “By coupling tightly to both the exocytotic and endocytic machineries,” they conclude, “voltage-gated Ca2+ channels are thus uniquely positioned to coordinate the SV recycling process.” Their model, however, is just a rough picture of a much more elaborate process scientists are just beginning to understand.
    --------------------------------------------------------------------------------
    1Yuan Chen et al., “Formation of an endophilin-Ca2+ Channel Complex Is Critical for Clathrin-Mediated Synaptic Vesicle Endocytosis,” Cell Vol 115, 37-48, 3 October 2003.
    2Two American scientists just received the Nobel Prize in chemistry October 8 for their work that revealed the structure and function of the water and ion channels in the cell membrane. See story in FoxNews.

    Life Found in the Genome Desert 10/16/2003
    Southwestern deserts are often filled with living things, if you look closely enough. Similarly, the “deserts” in the human genome, only sparsely populated with protein-coding genes, are turning up some surprising functions. Four California-based geneticists published a paper in Science Oct. 17 that found long-range enhancers in these regions:

    “Approximately 25% of the genome consists of gene-poor regions greater than 500 kb [kilobases], termed gene deserts. These segments have been minimally explored, and their functional significance remains elusive. One category of functional sequences postulated to lie in gene deserts is gene regulatory elements that have the ability to modulate gene expression over very long distances.”

    They found evidence that this is true, and scientists had better pay attention:

    “The demonstration that several of the enhancers characterized in this study reside in gene deserts highlights that these regions can indeed serve as reservoirs for sequence elements containing important functions. Moreover, our observations have implications for studies aiming to decipher the regulatory architecture of the human genome, as well as those exploring the functional impact of sequence variation. The size of genomic regions believed to be functionally linked to a particular gene may need to be expanded to take into account the possibility of essential regulatory sequences acting over near-megabase distances.”
    --------------------------------------------------------------------------------
    1Nobrega, Ovcharenko, Afzal, and Rubin, “Scanning Human Gene Deserts for Long-Range Enhancers,” Science 23 June 2003; accepted 8 September 2003, 10.1126/science.1088328.

    Ribosome Does Fast Forward Scanning 10/23/2003
    Remember cassette players that allowed you to scan ahead to the next song? Ribosomes in the cell are like tape readers that can translate one message, written in DNA, to another message, written in proteins. The “tape” that the ribosome reads is a string of messenger RNA, freshly delivered from the DNA code in the nucleus by other molecular machines. Once the tape is inserted into the ribosome, it reads the message and ties amino acids together to form a protein chain. If you watched the film Unlocking the Mystery of Life, you saw a computer animation of the process in slow motion. Now it appears that the ribosome has a scan function.
    Scientists have known about a puzzling phenomenon that occasionally occurs within the ribosome. For unknown reasons, the ribosome can disengage its reading head from the tape and fast-forward to another spot, then continue reading and translating at the next open reading frame (ORF). This is called “translational bypassing.” They know that this is signalled automatically by codes embedded in the messenger-RNA “tape”: a take-off code and a landing code, among others. What they didn’t know is whether the reading head continues to scan the code while disengaged. By analogy with cassette players, is it “fast forward” or “scan”? Apparently, it’s the latter.
    A team of scientists at the University of Washington figured this out by rigging two landing codes into the tape. They found that the ribosome always took the first one. This can only mean that the ribosome is able to scan the message while disengaged and detect the presence of the landing site. Their paper is published in the Oct. 23 online preprints of PNAS.1 Although it is still unclear why the ribosome would want to jump ahead on the recording, other researchers, like Raymond F. Gesteland, believe it is part of a “bag of tricks” the cell has to regulate gene expression or correct errors.
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    1Gallant et al., “Evidence that the bypassing ribosome travels through the coding gap,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.2233745100, Published online before print October 23, 2003.

    Time to Junk the Term “Junk DNA” 11/06/2003
    “Heirlooms in the Attic” is the way Mark Johnston and Gary Stormo describe, in the Nov. 7 issue of Science1, the potential discoveries awaiting scientists in the realms of non-coding DNA, disparagingly referred to as “junk DNA.” Genome research has understandably focused on genes that code for proteins, but what are these vast stretches of non-coding DNA? Are they junk or treasures?
    Hints that interesting gems are waiting to be uncovered include the fact that these areas are highly conserved,2 even more so, in some cases, than the genes. According to evolutionary thinking, if something is conserved, it must be important. Scientists are only beginning to uncover hidden functions in the non-coding regions. Do they regulate gene expression? Do they provide scaffolding or attachment points? “Uncovering the part that CNGs [conserved non-genic sequences] play in the cell will certainly require experimentation,” they say, “and that activity is likely to occupy many people for quite some time.” –
    Early in the Human Genome Project, people argued about what to sequence. Some advocated determining just the sequence of the protein-coding regions, because the vast majority of the genome is “junk” DNA. This would, they argued, be cost effective because most of the important information is in protein-coding DNA. Given what we’ve learned about the jewels in the genome’s attic, aren’t we glad they sequenced it all?
    See also Oct 16 headline.
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    1Mark Johnston and Gary D. Stormo, “Evolution: Heirlooms in the Attic,” Science Volume 302, Number 5647, Issue of 7 Nov 2003, pp. 997-999; 10.1126/science.1092271.
    2Dermitzakis et al., “Evolutionary Discrimination of Mammalian Conserved Non-Genic Sequences (CNGs),” Science. Originally published in Science Express as 10.1126/science.1087047 on October 2, 2003; Science, Vol. 302, Issue 5647, 1033-1035, November 7, 2003; 10.1126/science.1087047

    Intracellular Railroad Has Park-and-Ride System 12/04/2003
    Cells are like miniaturized cities, with elaborate transportation systems ferrying their cargo to and fro (see Feb. 25 headline). Just like a city may have railroads, busses, cars and monorails, the cell has multiple kinds of transport motors: dyneins, kinesins, and myosins. Scientists have learned that most of the roadways are like one-way monorails: actin filaments and microtubules, upon which the vehicles travel in one direction. But what if a passenger needs to jump from one system to another? ' No problem; the cell has mastered the art of ridesharing with its own park-and-ride system.
    In the Dec. 2 issue of Current Biology1, this is described by Marcus Maniak in a Dispatch entitled “A park-and-ride system for melanosomes.” Melanosomes are organelles (somes) that carry melanin, the pigment chemical that allows some organisms, including fish and amphibians, to change their skin color to match their surroundings. For this to work, the melanosomes need to hitch rides either to the exterior of the cell or the interior. He pulls together several recent findings to describe how this all works:

    “Together these findings suggested how melanosomes might move on actin filaments and showed that this type of motility is required for the even distribution of melanosomes within the cell. From these main observations, it became clear that, during aggregation, a cytoplasmic dynein motor carries melanosomes on the radially arranged microtubules towards the cell center (Figure 1B), while during dispersion a kinesin transports the granules to the periphery (Figure 1C), where they engage via a myosin V molecule with short actin filaments to be distributed further (Figure 1D). This switching of transport systems is a kind of miniature edition of modern urban traffic, where millions of workers leave the city centers in the evening on trains and board their cars at park-and-ride stations to complete their daily journey within the green peripheral belt.”

    As if that were not amazing enough, it appears that the drivers “talk” to each other with a communication system:

    “Although the work of Rodionov et al. has moved the field a large step further, there are obviously several issues that remain to be investigated. Exciting new findings addressing the coupling of motor molecules to the melanosome surface in other experimental animals open the possibility to speculate how the motors may talk to each other on a molecular level. At least for Xenopus there is now clear evidence that both dynein and kinesin couple to melanosomes via the dynactin complex. Moreover, both motors compete for the same protein component; this could allow one motor to gain access to the microtubule while the other is prevented from engaging successfully.”

    He describes how this “tug-of-war” competition is actually a kind of way for the motors to negotiate the right-of-way. Additional factors that attach to the vehicles or trackways may assist in making sure the rules of the road are obeyed. “Thus,” he concludes, “further exciting results are on the way to complete the picture of how melanosomes switch from one transport system to the other.”
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    1Marcus Maniak, “Dispatch: Organelle Transport: A Park-and-Ride System for Melanosomes,” Current Biology Vol 13, R917-R919, 2 December 2003.

    Elaborate Quality Control Governs the Cell’s Protein-Folding Factory 12/20/2003
    If it weren’t for quality control in our cells, we’d be dead. That’s the gist of an amazing Insight article in the Dec. 18 issue of Nature.1 “Aberrant proteins are extremely harmful to cells,” the authors begin. How harmful? Here is a short list of diseases that can result from improperly folded proteins or failures in the quality control systems that direct their formation: Creutzfeldt-Jakob disease, Alzheimer’s disease, and other degenerative diseases, scurvy, cystic fibrosis and more. In fact, serious defects in protein assembly are probably never seen, because they could prevent an organism from getting past the first cell division in the embryo. The only way a cell can live and grow is with the assistance of a host of traffic controllers, regulators, monitors, ushers, transporters, inspectors, security guards and emergency technicians maintaining the complex processes of protein assembly. Success must be ensured constantly, 24 x 7, that despite a flurry of activity, must maintain a state of dynamic equilibrium, called homeostasis.
    Each cell in the body is like a city of interrelated factories made up of protein machines and structures operating under strict regulations, built on coded instructions. One of the most important factories is the protein folding system, which ensures that newly-sequenced proteins coming out of ribosomes are folded into their correct (native) shapes. Proteins are made up of amino acids, usually hundreds of them, that are first sequentially assembled in ribosomes, based on templates sent from the DNA code. Then, they are folded into specific, complex three-dimensional shapes that perform numerous and diverse functions in the cell (see 06/13/02 and 05/31/02 headlines.) Protein folding is assisted by enzymes whimsically called chaperones (see 05/05/03 headline) but is also checked and rechecked by numerous other quality control systems (see 09/09/02 headline).
    In the current paper in Nature, the authors have unveiled more of the complexity in the quality controls governing protein folding. Some of the folding occurs in networked subway tunnels that run throughout the cell, called the endoplasmic reticulum (ER). Before getting into the ER, some proteins already begin their folding with assistance from certain chaperones. The authors explain, “In mammalian cells, proteins are translocated into the ER ... where they start to fold co-translationally [i.e., while they are en route into the ER]. Folding is completed post-translationally, and, generally, individual subunits have folded before assembly and oligomerization [the joining together of multiple chains] take place. Sequential interactions with distinct chaperones are required for each of these steps.” The job is completed inside the ER, and the finished protein “tool” is then sent on its way to work. But that is just the tip of a huge iceberg made up of a multitude of processes – hardware and software – that work together to ensure success.
    In the following examples from the article, entitled “Quality control in the endoplasmic reticulum protein factory,” don’t worry about unfamiliar technical terms. Just try to keep track of how many different players are involved in the team of factory workers dedicated to one job: folding a single protein. And keep in mind that each team player is itself a protein, built with the same quality control. You can almost envision little factory workers, each skilled at their specific tasks, alert and knowing just what to do, but it’s all done with chemicals! Be patient in these extended quotes, because the awe is in the details.

    1. Redox regulation: A sensitive chemical balance is maintained between reducing and oxidizing (redox) conditions along the protein’s pathway through the folding factory. “The redox gradient between the ER and the cytosol seems to be important for intercompartmental signalling, particularly in the integrated response to oxidative stress, in which adaptive responses emanating from different compartments are coordinated. And redox reactions with opposite electron fluxes must take place in the ER to mediate formation, isomerization and reduction of disulphides. The wealth of redox assistants allows these fluxes to be separate, and channels electron transport through specific protein–protein interactions.”
    2. Location specificity: “Although most folding factors in the ER are ubiquitously expressed throughout the body, some are tissue-type specific or cell-type specific, and probably fulfil a particular synthetic task.... For example, efficient collagen production requires the expression of hsp47, whereas a tissue-specific protein-disulphide-isomerase-like protein, PDIp, is produced in the pancreas and probably permits the massive secretion of digestive enzymes.”
    3. Bridge builders: Junctions called disulphide bridges are common in protein folds, and these, although weak, are carefully maintained by a host of enzymes called oxidoreductases: “The impressive number of oxidoreductases in the ER suggests that catalysis and regulation of disulphide-bond formation is crucial for folding. Energywise, in most cases, the contribution of a disulphide bond is hardly more than that of a single hydrogen bond [i.e., quite weak], yet, without disulphide bonds, native conformations are not obtained. Disulphide bonds cannot force a folding protein into a given conformation: in the sampling of conformations during folding in the ER, native and non-native disulphide cross-links are transiently formed [i.e., correct and incorrect links form and break easily, and must be guided]. Continuous activity of oxidoreductases probably ensures that these covalent links remain flexible until folding is completed.”
    4. Correct fold recognition: Even though a string of amino acids could conceivably fold in large number of ways, like a Rubik’s cube, somehow the chaperones are able to tell a correct (native) fold from an incorrect one. “Besides providing a unique folding environment, the ER has a crucial quality-control role. How does it discriminate between native and non-native proteins? The answer to this question depends primarily on ER chaperones. When folding or assembly intermediates expose hydrophobic [i.e., water-avoiding] surfaces, unpaired cysteines or immature glycans, ER-resident chaperones or oxidoreductases interact with them, and as a consequence they are retained in the ER or retrieved from the Golgi complex [see 11/12/01 headline]. By forming multimolecular complexes, folding factors in the ER may provide matrices that couple retention to folding and assembly. Immature proteins may also form aggregates that are excluded from vesicles exiting from the ER” [i.e., such that they are not ejected before they are ready].
    5. Fail-safe inspection: A protein needs to pass multiple layers of monitoring: “All proteins are subjected to a ‘primary’ quality control that monitors their architectural design through ubiquitous folding sensors (Table 1). ‘Secondary’ quality-control mechanisms rely instead on cell-specific factors and facilitate export of individual proteins or classes of proteins.... the ER is the main test bench where molecules destined for the extracellular space are scrutinized for their potential toxicity.”
    6. Feedback regulation: The ER not only does quality control, but sends messages back to the nucleus to regulate the production of more chaperones: “The reasons for having a quality-control system in the ER are easy to understand where protein folding and function are concerned, especially in multicellular organisms where development relies on the fidelity of protein secretion. Quality control can also regulate the transport or the activity of certain proteins during differentiation or in response to stress or metabolic requirements.”
    7. Waste control: When a protein cannot be folded after repeated attempts, more assistants are on hand to ensure proper dismantling and recycling: “Mutations or unbalanced subunit synthesis make folding or assembly — and hence exit from the ER — impossible. To maintain homeostasis [dynamic equilibrium], terminally misfolded molecules are ‘retrotranslocated’ or ‘dislocated’ across the ER membrane to be degraded by cytosolic proteasomes” [organelles equipped to break up badly-folded proteins and recycle their parts].
    8. Time limits: Somehow, the cell knows when a protein has had enough time to shape up or ship out: “A fascinating problem is how molecules that have not been given the time to fold (and therefore are unfolded) are discriminated from those that have failed to fold after many attempts (misfolded), and must therefore be disposed of. One way of timing glycoprotein quality control involves the sequential processing of N-glycans and in particular mannose trimming in the ER. It remains to be seen how substrates are eventually targeted to the retrotranslocation channels, how these are opened, and to what extent proteins must be unfolded to negotiate dislocation.”
    9. Workforce regulation: Like a company’s human resources department that responds to managers’ calls for more workers, the cell keeps track of how many chaperones are available, and sends out “help wanted” ads to the nucleus. “To maintain the efficiency of quality-control mechanisms in diverse physiological conditions, living cells have evolved [sic] regulatory circuits that monitor the levels of available chaperones. This is true for both the cytosol and the ER, and compartment-specific responses clearly exist that selectively restore optimal levels of the desired folding factors.”
    10. Emergency squads: The authors provide two examples of rapid-response traffic control teams: “The accumulation of aberrant proteins in the cytosol triggers the heat-shock response, resulting in de novo synthesis of hsp70 and other cytosolic chaperones. But if aberrant proteins accumulate in the ER, cells activate a different response, the unfolded protein response (UPR), which leads to the coordinated synthesis of ER-resident chaperones and enzymes.”
    11. Failure consequences: The authors give an example of what can go wrong when the system gets swamped, starved, or sent defective parts: “Physiologically, ER stress (a condition in which the folding machinery in the ER cannot cope with its protein load) can be caused by synthesis of mutated or orphan proteins, absence of cofactors (an example being scurvy, in which collagen cannot fold because of the lack of vitamin C), or a drastic increase in otherwise normal cargo proteins.”
    12. Unified response to varied inputs: A variety of signals can lead to the same Unfolded Protein Response (UPR) pathway: “How do the diverse unfolded or misfolded proteins that accumulate in the ER provoke the same pathway? The unifying concept is that BiP and other primary quality-control factors maintain the stress sensors in the ER in the inactive state, so that chaperone insufficiency triggers UPR whatever the nature of the cargo.”
    13. Meltdown regulation: What happens when the damage is so great, that further operation of the factory would be dangerous? Three independent controls make sure an orderly shutdown occurs (apoptosis, or cell death: see 04/09/02 headline). “The mammalian ER sensors, Ire1, PERK and ATF6, guarantee a tripartite response with synergic strategies. By phosphorylating eIF2alpha, PERK transiently attenuates translation, limiting protein load. ATF6 drives the transcriptional upregulation of many ER-resident proteins and folding assistants. Ire1 activates XBP-1, which in turn induces transcription of factors that facilitate ER-associated degradation (ERAD). The two-step activation of XBP-1 (transcriptionally induced by ATF6 and post-transcriptionally regulated by Ire1) guarantees the proper timing of the UPR [unfolded protein response] attempts to fold proteins precede the decision to degrade them. If the response fails to clear the ER, apoptosis is induced through several pathways.” The authors explain that “The UPR is multi-faceted and regulates proteins involved in quality control, ERAD and many aspects of the secretory pathway.”
    14. Balancing act: The quality control mechanisms walk a tightrope, with serious consequences for falling off: “Quality control must be a balance between retaining and degrading potentially harmful products and not preventing export of biologically active proteins. CFTR mutants in cystic fibrosis illustrate an overzealous quality control, where biologically active mutants cannot leave the ER. In this case, relaxing the quality control could cure the patient. But disease can also originate from defective degradation. If the rate of synthesis of a protein exceeds the combined rates of folding and degradation, a fraction of it will accumulate intracellularly.” Misfolded proteins have to make it across the ER membrane in time, and get degraded by the proteasome in time, or else aggregations (aggresomes) can build up inside or outside the ER. These are implicated in a number of “ER storage diseases.”

    The authors put this all into perspective: “Over the past few years, much has been learned about how proteins are handled by the ER folding and quality-control machineries, and some of this knowledge has begun to be translated to industry and to the clinic. Yet, many questions remain....” They hope that further elucidation of these complex, coordinated systems will allow drug designers to target faulty elements that cause degenerative diseases, or induce apoptosis in tumors to make them self-destruct. Clearly, though, in spite of the complexity already described, much remains to be learned about cell quality control.
    --------------------------------------------------------------------------------
    1Robert Sitia and Ineke Braakman, “Quality control in the endoplasmic reticulum protein factory,” Nature 426, 891 - 894 (18 December 2003); doi:10.1038/nature02262

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    Horizontal Gene Transfer More Widespread than Thought 07/10/2003
    A study of plant mitochondrial genomes published in the July 10 issue of Nature found five cases of horizontal gene transfer (HGT) between distantly related plants. This was unexpected, since HGT was thought to be only significant among bacteria and virtually absent from eukaryotes. They feel these discoveries are only the “tip of a large iceberg” that may cause major rethinking of the role of HGT not only in plants and mitochondrial DNA, but also in animals and in nuclear DNA. “Our findings raise many other questions,” say the authors, Bergthorsson, Adams, Thomason and Palmer, in “Widespread horizontal transfer of mitochondrial genes in flowering plants.”

    Bacterial Flagellum Rotation Speed Depends on Proton Flow 07/11/2003
    A bacterial motor responds to the fuel available. Howard Berg of Harvard, one of the world’s authorities on the bacterial flagellum, has established that there is a linear relation between proton motive force (pmf) and rotation speed. In a paper in the Proceedings of the National Academy of Sciences (July 11 online preprints), he says this was known for high speeds, but his present work establishes it for low speeds also.
    To measure these things, Berg took an E. coli bacterium with two flagella, and attached one to a small latex bead, and the other to a rigid surface. The first one could turn easily, but the other had a heavier load – the whole body of the bacterium. Plotting all the numbers, he found a linear relationship between pmf and rotation rate, from stalling speed up to 380 Hz at zero load. “The present work shows that a linear relation is true more generally,” he said, “providing an additional constraint on possible motor mechanisms.” He admitted in the discussion section that, “It is not yet known how the motor generates torque.”

    Simplest Protein a “Paradigm of Complexity” 07/15/2003
    Myoglobin (Mb), the substance that gives muscles their red color, was one of the first proteins studied. “Thirty years ago, ” state Frauenfelder, McMahon and Fenimore in a Commentary in the July 14 Proceedings of the National Academy of Sciences, “the textbook function of Mb, storage of dioxygen at the heme iron, was considered to be simple, fully understood, and consequently boring. Since then, the situation has changed: Mb is no longer fully understood.” Scientists are finding out that this single-chain (monomeric) protein, folded into an apparently shapeless blob, has multiple functions – and these derive from its ability to dynamically change shape:
    A protein does not exist in a unique conformation but can assume a very large number of somewhat different conformations or conformational substates. ... If a protein had just a single conformation, it could not function and would be dead like a stone.
    Proteins react to their environment, the pressure and temperature, and also to the atoms in their vicinity. In the case of myoglobin, oxygen and carbon monoxide molecules are able to cause it to open up, “as if the drawbridges ... were controlled from the outside of the castle!” they state with evident surprise. They conclude that this best-studied protein still sports some fundamental problems for biochemists and biophysicists to solve. What we are learning about its conformational motions during function makes it no longer boring! It symbolizes the beginning of discoveries that will undoubtedly be valid for all proteins. The authors call myoglobin the “hydrogen atom of biology,” analogous to the detailed model Bohr made of the simplest of atoms when he attempted to begin to understand the basic laws governing all atoms. As such, “The large number of substates and their organization and importance for function make Mb a paradigm of complexity.”

    Cell Translation Uses Rotating Locks and Keys 07/21/2003
    A French team has studied one of the molecules involved in the translation of DNA to protein, and found that it does some nifty shape changes when its accessory proteins are in place. The molecule is threonyl-tRNA synthetase, one of the family of 20 specialized molecules that attach the appropriate amino acid to its matching transfer-RNA (tRNA) carrier. The operation involves four parts: the synthetase, the tRNA, the amino acid threonine, and ATP. The abstract describes some of the activity observed:

    “The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8 angstroms at some C positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.”

    The paper by Moras et al. is published in the upcoming Journal of Molecular Biology, August 2003. (For a previous headline on the tRNA synthetase family, see June 9.)

    DNA End Capping More Complex Than Thought 07/25/2003
    An idea has been floating around for years to explain why cells grow old and die. Biochemists have known that DNA strands have end caps, called telomeres. These caps keep them from unwinding or sticking to other DNA strands, which, when it occurs, creates a crisis in the cell, and usually triggers cell death or apoptosis. Each time a cell divides, the story goes, it loses a telomere, because the duplication machinery could not get a grip on the last cap. This seemed to act like a countdown timer. When the telomeres hit zero, pop goes the apoptosis. An enzyme has been known, however, that repairs telomeres. Named telomerase, it was thought to work only in certain kinds of cells, and has been implicated in cancer. The idea was that out-of-control telomerase made cancer cells immortal when they should have died.
    Well, once again, the picture is more complicated than that. An international team has just reported in the journal Cell 07/25/2003 that “Telomerase Maintains Telomere Structure in Normal Human Cells.” They found that all cells express this repair enzyme, and that there is a complicated interplay between regulatory factors to keep a normal cell functioning through multiple cell divisions, with just the right number of telomeres for its needs and environment. Their observations “support the view that telomerase and telomere structure are dynamically regulated in normal human cells,” and that telomere length alone is not a sign of old age and impending death.
    Only when things go wrong with these regulatory mechanisms do cells either lose their last telomeres and die, or go wild into immortal replication cycles as in cancer. Telomerase is a key ingredient both in the regulation of cell proliferation and replicative lifespan, they found. Targeting telomerase in cancer treatment as a bad molecule may not be wise, therefore. It’s apparently a vital part of a normal cell’s operation. One thing is clear: “the relationships among telomere length, telomere expression, and replicative lifespan are more complex than previously believed.”

    Gates of the Membrane 08/06/2003
    A couple of papers in last week’s issue of Science reveal details of just two of nearly 360 specialized proteins in cell membranes that ferry necessary molecules across “the otherwise impermeable barrier imposed by the phospholipid bilayer.” They look like clever rockers forming a funnel on one side of the membrane. When the right molecule falls in, the funnel inverts and ejects the molecule onto the other side. These act as “molecular pumps, translocating their substrates across membranes against a concentration gradient; this thermodynamically unfavorable process is powered by coupling to a second, energetically favorable process such as ATP hydrolysis or the movement of a second solute down a transmembrane concentration gradient.” The two studied here, LacY and GlpT, use the latter method.

    Nanocells are Naah, No Cells 08/09/2003
    Earlier claims that nanobacteria exist, tiny cells an order of magnitude smaller than the smallest known cells, are apparently unfounded. Nature Science Update reports on a paper in Geology Aug. 2003 that the alleged fossils of nanobacteria appear to be by-products of enzyme-driven tissue decay; i.e., just clumps of leftover digested material from larger living things.

    Beautiful: The Maximum Output from Minimal Cells 08/13/2003
    Dry science journals do not often talk about beauty, but Donald A. Bryant (Penn State) entitled his Commentary in the Proceedings of the National Academy of Sciences (online preprints, 08/13/03), “The beauty in small things revealed.” There is a tiny, minimalist cyanobacterium in the oceans that is so plentiful in numbers, it and one other species might account for as much as two-thirds the total CO2 fixation in the oceans, and one-third the primary biomass production on earth. This makes it a key player in the global carbon cycle. “The contribution of marine photosynthesis to the global carbon cycle was grossly underestimated until recently,” Bryant comments. “...As every microbiologist inherently knows, little things can be the cause of much greater things that are often of utmost importance, and this is especially true of phytoplankton.” Yet this key player was only discovered 15 years ago.
    Bryant refers to another paper in the same issue of PNAS by Dufresne et al., who sequenced the genome of this organism named Prochlorococcus marinus. They found it to be very near the theoretical lower limit in size for an autotrophic (self-feeding) photosynthetic organism, one ten-millionth of a cubic meter. “Because of its remarkable compactness,” they write, “the genome of P. marinus SS120 might approximate the minimal gene complement of a photosynthetic organism.” Some of its systems – DNA repair, chaperones, transport systems, motility, and nitrogen metabolism among them – are scaled down from other, larger bacterial cells. It also lacks duplicate genes for photosystem II components (although it has the complete set). But it has enough genetic information and synthetic machinery to make all its own nutrients with sunlight. This is a non-trivial toolkit: “it must have the ability to synthesize all cellular constituents, including amino acids, nucleotides, coenzymes, etc. from CO2 and mineral salts.” The small size of Prochlorococcus also has the advantage of a greater surface-to-volume ratio, less self-shading, and more efficient light capture. “Being minimalistic,” Bryant says, “does not necessarily mean that Prochlorococcus sp. is less competitive.” The little cells can diversify and adapt well. “Yes,” he concludes, “small things can be simple and yet highly successful on a global scale.” There is a lay summary of the article on Nature Science Update by John Whitfield.

    Understanding Cells: Think Information, Logic Circuits 08/21/2003
    The Concepts article in Nature 08/21/2003 is about “Systems biology: Understanding Cells” by Paul Nurse. A striking feature of his article is the repeated use of the word information:

    “Many of the properties that characterize living organisms are also exhibited by individual cells. These include communication, homeostasis, spatial and temporal organization, reproduction, and adaptation to external stimuli. Biological explanations of these complex phenomena are often based on the logical and informational processes that underpin the mechanisms involved....
    Most experimental investigations of cells, however, do not readily yield such explanations, because they usually put greater emphasis on molecular and biochemical descriptions of phenomena. To explain logical and informational processes on a cellular level, therefore, we need to devise new ways to obtain and analyse data, particularly those generated by genomic and post-genomic studies.
    An important part of the search for such explanations is the identification, characterization and classification of the logical and informational modules that operate in cells. For example, the types of modules that may be involved in the dynamics of intracellular communication include feedback loops, switches, timers, oscillators and amplifiers. Many of these could be similar in formal structure to those already studied in the development of machine theory, computing and electronic circuitry.”

    Nurse identifies three types of information seen in cells: sequence data, interaction data, and functional data. He feels that this logical, informational approach to the study of cells will be more productive than just studying the individual molecules in detail:

    “A useful analogy of what is being proposed is the analysis of an electronic circuit. Once the detailed operations of different types of electronic components have been identified, it is possible to gain insight into what an electronic circuit can do simply by knowing what components are present and how they are connected, even if their precise dynamic behaviour has not been determined. The various logical and informational modules implicated in a biological phenomenon of interest have to be integrated in order to generate a better understanding of how cells work.”

    Paul Nurse feels that this information-theoretic approach to the cell could generate a great deal of experimental work. “The identification and characterization of these modules will require extensive experimental investigation, followed by realistic modelling of the processes involved,” he predicts. “Such analyses would allow a catalogue of the module types that operate in cells to be assembled.” But this approach will work only if there is a finite set of such modules:

    “The success of this general approach depends on there being a limited set of biochemical activities and molecular interactions that together can solve the myriad logical and informational problems found in biological systems. If there is only a restricted set of processes that are efficient and stable in operation and which have been exploited by evolution [sic], then there should be only a limited set of possible solutions to real biological problems. Of course, if nature shows no such restraint [sic], then we must go back to the drawing-board if we are ever to understand its complexity.”

    Paul Nurse is at the Cell Cycle Laboratory, Cancer Research UK, Lincoln’s Inn Fields, London.

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    Your Body Has Transistors Superior to Intel’s 05/01/2003
    Roderick MacKinnon’s team has done it again. The headline at Rockefeller University proclaims, “Voltage-dependent channel structure reveals masterpiece responsible for all nerve, muscle activity.” Building on the art metaphor, the news release begins, “Scientists studying the tiny devices — called voltage-dependent ion channels — that are responsible for all nerve and muscle signals in living organisms for 50 years have been working like a bunch of blindfolded art critics. ... Rockefeller’s Roderick MacKinnon, M.D., a Howard Hughes Medical Institute Investigator, Youxing Jiang, Ph.D., and their colleagues have removed the blindfold to reveal a masterpiece of nature’s engineering” :
    The masterpiece is an exquisite voltage-regulated pore in the cell membrane that attracts and transmits potassium ions, maintaining an electrical potential with performance specs superior to man-made transistors. The team found that the channel operates with four charge-sensitive protein paddles around the periphery of the channel. They open to permit the correct ions through, and close to adjust the voltage. Proper voltage is maintained via a feedback loop that is sensitive to changing conditions in the environment. Their experimental results made the cover story of the May 1 issue of Nature.
    Potassium channels are vital to muscle and nerve activity, and are highly conserved in all organisms, from the Archaea living around hydrothermal vents at the bottom of the ocean, to the human gymnast on the high bar. The importance of these molecule-size gates is summed up in the news release: “The entire sequence of events takes only a few milliseconds, and occurs tens of thousands of times every day in human beings and organisms of all varieties. Without this hair trigger electrical system, life would be more than calm. There would be scant possibility of thinking, breathing or movement.”

    Are Germs Good Bugs Gone Bad? The Case of Anthrax 05/01/2003
    Now that the anthrax genome has been decoded, scientists are surprised that most of it looks like another milder bacterium, Bacillus cereus. Only about 3% of their genomes are significantly different. In addition, the pathogenic genes are not in the nucleus, but in plasmids (smaller, circular strands of DNA in the cytoplasm). They give the appearance of having been imported by horizontal gene transfer. One of the papers in Nature suggests that both bacteria acquired toxic elements from soil bacteria in this manner, and “Other major differences between B. anthracis and B. cereus may have been effected through altered gene expression rather than loss or gain of genes.” Some are wondering if anthrax acquired its toxicity recently. One team asks, “Findings from this genome sequence analysis raise further questions about the biology of B. anthracis; for instance, what are the roles of putative ‘virulence’ genes in close relatives of B. anthracis that do not cause anthrax, and do they actually contribute to virulence in B. anthracis?
    EurekAlert summarizes two papers in the May 1 issue of Nature that report the decoding of the anthrax genome.

    Not All Pseudogenes are Pseudo Genes 05/01/2003
    At least one pseudogene has a function, claims a team scientists from Japan and UC San Diego (see UCSD Health Science News). Long assumed to be dead copies of true genes that are devolving into useless relics, pseudogenes, which are common in eukaryotic DNA, may not be so useless after all. The team found a pseudogene that, while not coding for a protein, affects the expression of the true gene. The pseudogene apparently stabilizes the expression of a similar protein-coding gene on another chromosome. Without the pseudogene, lab mice developed abnormal kidneys and bones. The team discovered the function of the pseudogene by accident, reports SciNews, as they were preparing the mice for a different experiment. They suspect similar mechanisms may be at work in most other pseudogenes, and “hope to show that pseudogene-gene interaction is a general mechanism taking place in many cellular interactions.” Their technical paper is published in the May 1 issue of Nature Science Now explains that pseudogenes may help keep normal copies functioning.

    Protein Has Its Own Private Dressing Room 05/05/2003
    Any star of stage and screen has her own private dressing room, and so does the star of cellular activity, the protein. But the protein’s dressing room would make the actress envious; it has a powered double door.
    In the May 2 issue of Cell, a team of Stanford scientists studied the ATP-powered lid on one of these dressing rooms, called chaperonins, or chaperones from their role as supervisors of protein folding. Chaperonins are large, complex proteins shaped somewhat like a barrel with a lid. When a newly-joined chain of amino acids comes off the ribosome assembly line, it is subject to damage from the beehive of activity going on in the cytoplasm. It needs a quiet place to fold. The chaperone lid opens, the polypeptide enters, the lid closes, and safe inside, the chain collapses into its precise shape it needs to function. Then the lid opens and the protein exits, ready to go on-stage.
    Any action in the cell needs power. The lid on the chaperone is powered by ATP, the common energy currency in the cell. But ATP alone was not enough; As near as these scientists could tell, the camshaft (gamma subunit) on the ATP synthase motor is what triggers the lid to close. The Stanford team found that prokaryotic chaperones have lids that snap on from the outside, but eukaryotic chaperones have a built-in lid. In either case, the lid closing encapsulates the protein chain inside, and is essential for the chain to complete its folding operation; chaperones without lids could not produce folded proteins. Instead, like an actor getting the hook, a guard named protease takes the misfolded protein to the unemployment desk.
    Some proteins, like actin, require the help of this secure room to fold; others fold spontaneously inside. The next turn of the cycle opens the door, and out pops the protein for the production, complete with hook resistance.

    Enzyme Speeds Up Slow Reaction by 10exp21 05/06/2003
    The slowest known biological reaction would take a trillion years on its own, but an enzyme does it in 10 thousandths of a second. Richard Wolfenden of the University of North Carolina discovered this amazing fact by studying the reaction time of phosphate monoesters that are commonly used in cell signaling. The earlier record he had published in 1998 was 78 million years for an uncatalyzed biological transformation that is “absolutely essential” for creating the building blocks of DNA and RNA. This new record means that you could only expect the reaction to occur once in 100 times the assumed lifetime of the universe without the help of the enzyme. Paraphrasing Wolfenden, “that information would allow biologists to appreciate what natural selection has accomplished over the millennia in the evolution of enzymes as prolific catalysts,” says EurekAlert where this story can be found.

    How a Mosquito Became Insecticide Resistant 05/08/2003
    A French team publishing in the May 8 issue of Nature studied why disease-carrying mosquitoes became resistant to insecticides. It was due to “a loss of sensitivity of the insect’s acetylcholinesterase enzyme to organophosphates and carbamates” that are ingredients of the pesticides. In some cases a single point mutation conferred the resistance.

    Automatic Bandages in 10 Seconds 05/08/2003
    The gym class may have a first aid kit with Ace bandages, gauze and adhesive pads, but at the cellular scale, the first aid is automatic. In the May 8 issue of Nature, Juliet A. Ellis from King’s College, London, describes how your body has a fast-acting, automatic bandaging system:

    “Cell membranes in tissues such as skin, gut and muscle are routinely exposed to mechanical damage, which can produce holes in them. When that damage is not repaired, the consequences can be severe - often resulting in cell death - and may contribute to the development of the muscle degenerative diseases termed muscular dystrophies. From a combination of observations on human muscular dystrophy patients and experiments with mice, Bansal et al. (page 168 of this issue) now report that a protein called dysferlin is a component of the mechanism for resealing the holes, and thus healing the muscle membrane.
    Membrane resealing is generally carried out by a mechanism that resembles the calcium-regulated release of vesicles from a cell (exocytosis). The repair pathway is initiated by an influx of calcium through a wound, resulting in an increase in calcium levels at the site of injury. This, in turn, triggers the accumulation of vesicles, which fuse with one another and then with the plasma membrane, within the injury. A 'patch' is thereby added across the wounded area, resealing the plasma membrane. The entire process - which remains largely mysterious - takes between ten and thirty seconds.”

    For this to work, the cell needs several coordinated mechanisms: a way to sense the damage, a way to signal the repair team, the materials available to make the patch, and procedures for applying the patch and closing out the alarm.

    Treasure Found in DNA Junkyard 05/23/2003
    “Not Junk After All,” says Wojciech Makalowski of so-called “junk DNA” (a term coined by the late Sozumu Ohno to describe apparently useless, repetitive sequences in the genome that do not code for genes). Writing in the May 23 issue of Science, he says the junkyard was really a treasure mine :

    Although catchy, the term “junk DNA” for many years repelled mainstream researchers from studying noncoding DNA. Who, except a small number of genomic clochards, would like to dig through genomic garbage? However, in science as in normal life, there are some clochards who, at the risk of being ridiculed, explore unpopular territories. Because of them, the view of junk DNA, especially repetitive elements, began to change in the early 1990s. Now, more and more biologists regard repetitive elements as a genomic treasure.
    How do the mislabeled pieces of junk shine like gems? They apparently regulate the expression of gene-coding regions through alternative splicing. Describing an example published by an Israeli team in the same issue of Science, Makalowski explains that the alternative splicing involves an interplay with the giant molecular machine called the spliceosome, and is finely tuned:

    "It is even more tricky to maintain the delicate balance of signals that cause an exon to be spliced alternatively--you make one mistake (a point mutation) and either a splicing signal becomes too strong and an exon is spliced constitutively, or the signal becomes too weak and an exon is skipped."

    Makalowski thinks the additional copies of genes allow one to be preserved and the other to be a source of evolutionary novelty.

    “Unfortunately, most mutations will lead to abnormal proteins and are likely to result in disease. Yet a small number may create an evolutionary novelty, and nature’s “alternative splicing approach” guarantees that such a novelty may be tested while the original protein stays intact.
    Another way to exploit an evolutionary novelty without disturbing the function of the original protein is gene duplication (see the figure). Gene duplication is one of the major ways in which organisms can generate new genes. After a gene duplication, one copy maintains its original function whereas the other is free to evolve and can be used for “nature’s experiments.”
    He realizes he is sounding anthropomorphic, but sheepishly continues his analogy in the concluding sentences (emphasis added):

    "These two papers demonstrate that repetitive elements are not useless junk DNA but rather are important, integral components of eukaryotic genomes. Risking personification of biological processes, we can say that evolution is too wise [sic] to waste this valuable information. Therefore, repetitive DNA should be called not junk DNA but a genomic scrap yard, because it is a reservoir of ready-to-use segments for nature’s evolutionary experiments."

    The other paper he refers to was a study by Iwashita et al a few years ago that suggested transposable elements permit a kind of modular programming. A cow was found to have two copies of a gene, but one copy had an inserted endonuclease module. He concluded that this arrangement allowed it to evolve a new function while the other copy without the module maintained the cow’s original fitness.

    Factoid: The Nuclear Pore Complex 06/02/2003
    Impress your friends today: tell them about Nuclear Pore Complexes. These are elaborate, specialized pores in the nuclear membranes that surround the nucleus of each cell in your body like a skin. The pores look something like complex basketball hoops with rings and studs that act like electronic gates. Their job is to control traffic in and out of the nucleus. Each nuclear pore complex works so fast, it can authenticate somewhere between 520 and 1000 pieces of cargo per second. A typical nucleus has about 2000 to 4000 or more of these gates, which are made up of 30 or more very complex proteins. They all have to be disassembled and reassembled every time a cell divides. (Believe it or not, this is a vastly oversimplified summary of a much more complicated picture.)
    Source: Developmental Cell, June 2, 2003, review article by Suntharalingam and Wente.

    Germs For Your Health 06/04/2003
    “Most people’s views of bacteria are of menacing, disease-producing entities. Au contraire,” says Jeffrey I Gordon of Washington University School of Medicine (St. Louis), quoted in Science News 163:22, p. 344. “I think that most of our encounters with bacteria are mutually beneficial, friendly, and part of our normal biology. .... They’ve insinuated themselves into our biology and coevolved with us.”
    The article by John Travis lists several ways our intestinal flora help us. They break down complex sugars, signal the gut lining to stimulate defenses against pathogens, help the gut mature, and help it detoxify compounds. One kind is mostly active during lactation to help an infant digest complex sugars in the mother’s milk – in fact, the Nestle company farms this bacterium and incorporates into some of its infant formula and yogurt “to promote gastrointestinal health.” Scientists have found that rodents raised without a certain bacterium must consume about 30 percent more calories to maintain their body weight; this means the bacterium helps a mammal to digest its food. Other microbes stimulate our own cells to put up an “electric fence” to keep out harmful germs, but are not affected themselves. In return, the friendly bacteria get to feed off leftovers. There may be 1,000 different kinds of bacteria living in our intestines. Scientists have barely begun to explore the variety of these organisms, which according to estimates “may together possess as many unique genes as a person does, and perhaps far more.” Your little passengers “outnumber all the cells in your body, perhaps by as much as a factor of 10.”

    How to Tweak a Translator 06/09/2003
    As discussed here several times before (April 29, Nov. 1), DNA translation depends on a family of 20 specialized proteins that act as language interpreters. They are called the aminoacyl-tRNA synthetases (aaRS), and their unique property of being able to precisely match an amino acid to the transfer RNA that codes for it means they understand two codes: the nucleotide code of DNA, and the amino-acid code of proteins. How could such an interpreter evolve?
    In the June 9 online preprints of the Proceedings of the National Academy of Sciences, three French biochemists claim to have fused parts together to create an “artificial” tRNA synthetase. Each aaRS molecule has four functional parts: a domain that binds AMP to the amino acid, a domain that acylates the amino acid, a domain that edits the tRNA attachment, and a domain that joins the two together. The ability of each of these functions to work depends on the precise order of the amino acids in the synthetase (for alanine’s synthetase, 876 of them). These scientists took out the normal part of amino acids #368-461, the part involved in aminoacylation, and fused in some of their own polypeptides they had selected for their ability to acylate the amino acid alanine. Out of seven mutants, some did better than others, and none showed any significant energy penalty in the other end’s ability to bind RNA.
    They also tried substituting other amino acids in the active site of their “artificial” synthetase. Each substitution reduced the ability to acylate alanine, some 2-fold, some 5-fold and with three changes, 10-fold. Each mutant also lost ability to act specifically on alanine. There did not seem to be much tolerance, therefore, for changes in a 10-peptide sequence located in the heart of the active site.
    What do they make of this? In the discussion, they feel they have demonstrated that fusing a replacement string into part of the synthetase did not destroy its ability to do its other functions. “Importantly,” they say,

    “the two components, adenylate synthesis and specific RNA binding, were generated independently. ... Thus, the results are consistent with the idea that early tRNA synthetases arose from small, idiosyncratic RNA-binding elements being fused to domains for adenylate synthesis. These RNA-binding elements might have developed originally to bind and protect ribozymes (to give early ribonucleopeptides or ribonucleoproteins; refs 44-47). The fusions of RNA-binding peptides to domains for adenylate synthesis may have been the first step in developing protein-based synthetases that overcame the ribozyme-based system of aminoacylation.”

    Since the cost to the RNA-binding portion was inconsequential, they feel the two main functions of the synthetase could have arisen independently, and serendipitously come together to take over the job ribozymes were doing [i.e., in the hypothetical RNA World scenario].

    Picture of Protein Evolution Emerging? 06/16/2003
    “Most proteins have been formed by gene duplication, recombination, and divergence,” declare scientists from Cambridge and Stanford in the June 13 issue of Science. “Proteins of known structure can be matched to about 50% of genome sequences, and these data provide a quantitative description and can suggest hypotheses about the origins of these processes.” With growing numbers of genomes decoded, they feel we are well on the way to answering fundamental questions about how the huge assortment of proteins arose:

    “During the course of evolution, forms of life with increasing complexity have arisen. What are the mechanisms that have produced the increases in protein repertoires that underlie the evolution of more complex forms of life? How are proteins organized to form pathways? Answers to such questions at the molecular level began to appear 40 years ago, but it is only with the advent of complete genome sequences that we have begun to get a comprehensive view.”

    “At present,” they admit, only “close to 50% of the sequences in the currently known genomes are homologous to proteins of known structure,” yet “this half of the protein repertoire have given us a detailed picture of its evolution.” They discuss how proteins fall into domains, and these are organized into families that seem to obey a power-law distribution; i.e, “A few families have many members and many families have a few members.” Even proteins with different sequences can often be matched with others possessing similar structure. Many of these are paired with other domains. Of all the million-plus possible pairs of known families, only a few thousand are used. This, they feel, is evidence of selection for function. Also, the fact that “combinations of particular pairs of domains are found in only one sequential order ... suggests that conservation of sequential order in domain combinations is usually found because the combinations descend from a common ancestor.”
    The authors feel confident that we understand the basics of how new complexity arises from the protein pool:

    “It is now clear that the dominant mechanisms that produce increases in protein repertoires are (i) duplication of sequences that code for one or more domains; (ii) divergence of the duplicated sequences by mutations, deletions, and insertions to produce modified structures that may have useful new properties and be selected; and, in some cases, (iii) recombination of genes that results in novel arrangements of domains.”

    But how would metabolic pathways arise? They introduce the problem: “Proteins do not function by themselves but as part of an intricate network of physical complexes and pathways. How does the duplication, divergence, and recombination process fit into the formation or extension of pathways?” They propose that mutated proteins might either be recruited to new substrates within existing pathways, or jump to different pathways. They observe, “An examination of the functions of the members of different families of domains shows that, nearly always, it is the catalytic mechanism or cofactor-binding properties that are conserved or slightly modified and the substrate specificity that is changed. This suggests that it is much easier to evolve new binding sites than new catalytic mechanisms.” This tends to scramble the evolutionary picture, though: “This has led to a mosaic pattern of protein families with little or no coherence in the evolutionary relationships in different parts of the network.” Can the evolutionary history be seen by comparing unrelated organisms, then?

    “The comparison of enzymes in the same pathway in different organisms also shows that proteins responsible for the particular functions can belong to unrelated protein families. This phenomenon is called “nonorthologous displacement”. Variations come not just from changes in specific enzymes. In some organisms, sections of the standard pathway are not found and the gaps are bypassed through the use of alternative pathways. Together, these variations produce widespread plasticity in the pathways that are found in different organisms....

    One final question remains: how did the first proteins originate? And are new ones originating now?

    “The earliest evolution of the protein repertoire must have involved the ab initio [Lat., from the beginning] invention of new proteins. At a very low level, this may still take place. But it is clear that the dominant mechanisms for expansion of the protein repertoire, in biology as we now know it, are gene duplication, divergence, and recombination. Why have these mechanisms replaced ab initio invention? Two plausible causes, which complement each other, can be put forward. First, once a set of domains whose functions are varied enough to support a basic form of life had been created, it was much faster to produce new proteins with different functions by duplication, divergence, and recombination. Second, once the error-correction procedures now present in DNA replication and protein synthesis were developed, they made the ab initio invention of proteins a process that is too difficult to be useful.”

    In conclusion, they remind the reader that genome size is not the measure of complexity (rice has more genes than people); instead, “complexity does seem to be related to expansions in particular families that underlie the more complex forms of life.” So the key to understanding the evolution of the protein repertoire will be to compare how families of proteins in diverse organisms have been duplicated and recombined.

    Bacteria More Orderly Than Previously Known 06/17/2003
    Bacteria are not simple bags of protoplasm. Since they lack the organelles and nuclei that eukaryotic cells possess, scientists used to think their contents were fairly unstructured and homogeneous. That view is changing, say Zemer Gitai and Lucy Shapiro in the June 16 online preprints of the Proceedings of the National Academy of Sciences. “Historically,” they agree, “perhaps because of their general lack of compartmentalized organelles, bacteria were viewed as relatively uniform at the subcellular level.” New microscopic techniques are unveiling highly ordered structures, like protein spirals and rings that oscillate between the poles and allow the cell to locate the midpoint for cell division. “Perhaps the most important lesson to be learned from the work by Shih et al,” (who imaged the spiral proteins) “is that the more closely we look, the more order we see within bacterial cells. The fact that the phrase ‘bacteria are not just small bags of enzymes’ has become cliché is a sign that bacterial cell biology is coming of age.”

    For a related story, see our Jan 16 headline about spiral action of the bacterial cytoskeleton that repairs the inner cell wall.

    Surprise: Y Chromosome Protects Itself with Palindromes 06/18/2003
    Cheer up, men: your Y chromosome is not going extinct. Since the Y has no backup copy, geneticists thought it might mutate itself into useless junk in just 10 million years. Well, the Y chromosome map has just been completed, reports Nature Science Update, and of all the clever things, the Y has built-in self-defense in the form of palindromes. Just like the phrase “Madam, I’m Adam” can be read the same backwards and forwards, there are large gene-coding regions on the Y that can be decoded in either direction. The article explains:

    “These palindromes house many genes - which means that there is a copy at each end of the palindromic sequence. These provide back-ups should harmful mutations arise. The mirror-image structure also allows the arms to swap position when DNA divides. Genes are shuffled and bad copies are purged.”

    David Page at MIT remarked, “The Y chromosome is a hall of mirrors.” More surprises are expected now that the full map of the chromosome has been published (it’s the cover story of Nature June 19). Now that the male chromosome “reveals that we have underestimated its powers of self-preservation,” maybe men will finally start getting some respect.
    “Male chromosome full of surprises,” is the way Science Now entitled their summary of the findings. The Y is not a graveyard of genes, nor a shriveled up remnant of the larger X chromosome. Its new-found capabilities, dynamically shuffling its genes to weed out defects, has given scientists a new appreciation for it. As one researcher put it, this has “brought a lot of honor to males.”

    Scientists Watch Motors Unwind DNA 06/19/2003
    Andrew Taylor and Gerald Smith from Fred Hutchinson Cancer Research Center (Seattle, WA) announced in Nature June 19 that “RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity.” In the same issue, Mark S. Dillingham, Maria Spies and Stephen C. Kowalczykowski of U.C. Davis came to a similar conclusion. Working independently, these teams watched an important molecular motor in action and determined that it is two motors in one, with a slow motor and fast motor working side by side on the same track. How can that be, and why?
    RecBCD helicase is the molecular machine that travels along a DNA double helix, unwinds it, and separates the strands so that the translation machinery can get to it. This combination enzyme (RecB + RecC + RecD) is a member of a superfamily of helicases, or enzymes able to unwind and separate DNA. Simpler helicases separate the two DNA strands into a Y-like tail, but RecBCD has the unusual property of creating a loose tail on the RecD side and a loop and a short tail on the RecB side (RecC, not a motor, appears to help RecB in its action). Combined, RecBCD is among the fastest of helicases: it can cover 370 base pairs per second, according to Taylor and Smith, or up to 1000 base pairs per second, according to Kowalczykowski et al.
    Both the RecB and RecD motors can travel along DNA separately, but are polar opposites: one moves along one strand, one along the other. Of the two, RecD is the speed demon; RecBC only moves 20% as fast. The motors are not nearly as fast or stable acting alone. Separately, they fall off the track after 50 base pairs, but together, can cover 400-600 times as much ground: 20,000 (Taylor and Smith) or 30,000 (Kowalczykowski) at full speed.
    So why two engines in this race car? Taylor and Smith suggest that it adds stability; a motor is less likely to fall off the DNA track when combined with another, but why the speed difference? This will take more study. All they can conclude is, “This asymmetric feature might impart RecBCD enzyme’s asymmetry in other aspects of its promotion of genetic recombination.”

    Cell to Phagocyte: I’m Dying – Eat Me 06/27/2003
    Cells go the way of all the earth, but their society cleans up after them. This occurs through an elaborate signalling procedure that biochemists are beginning to uncover, as explained in a Minireview in Cell, June 27 by Kodi S Ravichandran (Univ. of Virginia). A cell undergoing death throes by caspase activation (in itself an elaborate shutdown process) sends out “eat me” signals that are recognized by the roving clean-up squad, the phagocytes. Normally, a cell wears a “Don’t eat me” tag, but this is removed and a phosphatidylserine (PS) tag pops up on the outer membrane. Simultaneously, LPC and/or other signals are secreted in search of a nearby phagocyte, with a “silent invitation to dinner.” The dying cell wears the Eat-Me signals on its outer membrane. An approaching phagocyte turns on anti-inflammation signals, as if to say to others nearby, “Nothing to get inflamed about; I can handle this one.” After engulfing the dying cell, it re-arms the inflammation alarm.
    Through this system, needless inflammation is avoided, and the streets and alleys are kept clear of cellular corpses. The author summarize, “An evolutionarily conserved machinery exists for engulfment of apoptotic cells from worm to mammals.”
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    Gatekeepers of the Cell Nucleus Revealed 03/04/2003
    Five thousand gates control access in and out of the cell nucleus: the Nuclear Pore Complexes (NPCs). In the March 4 issue of Current Biology, two Canadian biochemists survey what is known about them. On a molecular scale, they are huge assemblages with many parts, made up of 30 different types of proteins. But these are not just holes in the nucleus; they are departments of homeland security. Squads of other proteins scan the visitors and badge them if authorized so that they can run the gauntlet. And that’s not all. Now evidence is growing that the NPC, through its power to control what enters and exits the nucleus, is a regulator of gene expression. The authors say that eukaryotes (that’s us and other multicellular organisms) have the “Cadillac” model NPC composed of 125 million atomic mass units; single-cell organisms like yeast, with 50 million, have the “sportier” version.

    Ready-Mix Patch Kit Stands Ready To Repair Your Body’s Brick Walls 03/19/2003
    You’ve probably used those packets with two compartments that do something when the dividing membrane is broken, allowing the components to mix: instant heat, instant cold, instant glue, or instant light. Your body has something like that to repair its tissues. Tissues are the webs of specialized cells that distinguish us multicellular organisms from the rest, and the bulk of tissues are composed of epithelium. Epithelial cells line up in tightly-knit ranks forming the lining of most organs, the lungs and windpipe, the digestive tract, and the skin. Because they are subject to injury, these membranes must have a means of repairing themselves quickly. So they have a kind of ready-mix patch that works only when two components combine. But the system must work flawlessly, or a disaster can result.

    Keith Mostov and Mirjam Zegers talk about this in the Mar. 20 issue of Nature, “Cell Biology: Just Mix and Patch,” reporting on work by Paola Vermeer and company in the same issue. Epithelial cells have two linings. Consider the respiratory tract as an example. One lining, the apical side, faces the airway. The other, the basolateral side, lines the other end and the neighboring cells. These two linings are segregated by a kind of O-ring seal that makes a tight fit between neighboring cells. Scientists recently found that the basolateral membrane has one component of the patch, called erbB2, and the apical side has a matching component called heregulin. Normally kept apart, they can be brought in contact when a breach occurs in the epithelial tissue. Together, they activate a complex series of steps leading to cell division and presto! the gap is filled in with another snug-fitting cell, and life goes on. It is essential these active ingredients don’t mix at the wrong time. Too much cell division and you know what happens — cancer. Science Now has a news write-up on this story, and its discovery that is “so beautifully simple.”

    A few more Cool Cell Tricks were reported recently:

    • Cells have an exquisite toolkit for dealing with iron. Three New Zealand scientists writing a Perspective special feature in the Proceedings of the National Academy of Sciences describe a family of proteins called transferrins that clamp around iron and delicately transport this very toxic atom to wherever it’s needed in the cell. The clamp has a hinge that opens the structure and disgorges the iron when it is safe to do so. Another protein called hemopexin transports heme by holding it in the center of a four-part structure.
    • Another Special Feature in the same issue talks about nitrogenase, which we discussed Sept. 6, 2002. Two Harvard chemists attack this puzzling molecule with the zeal of Captain Ahab pursuing Moby Dick (this is actually how they end their article), but in spite of the best efforts of scientists for decades, “Few problems in bioinorganic chemistry have proved as challenging and refractory.” They speak of techniques this molecule uses that are “biologically and chemically unprecedented,” and marvel like Scotty and Captain Kirk aboard an alien ship trying to figure out a novel dilithium crystal reactor. Hidden inside the inner sanctum of this molecular machine is a secret method for separating nitrogen atoms at room temperature that is the dream of agricultural chemists, because artificial nitrogen fixation (e.g., fertilizer making) is costly and energy intensive. “The synthetic problem of nitrogenase, nevertheless, remains unsolved,” but they think we’re getting warmer.
    Current Biology for March 18 has a quick guide to a very versatile gene called APC (adenomatous polyposis coli), without which we either die or get colon cancer. It moves all over the cell, in and out of the nucleus, even riding the intracellular railroad. APC has many jobs. It’s a potent tumor suppressor, it regulates gene transcription, and it has a role in “maintaining adherens junctions, and also helps to tether mitotic spindles to the cortex and to orient them in the epithelial plane. In mammalian cells, APC has been implicated in cell migration. APC also helps safeguard the fidelity of chromosome segregation in mitotic cells.”

    DNA Repairmen Can Back Each Other Up 03/21/2003
    The DNA Damage Response team has many specialized technicians, but now scientists have found some of them can fill in for a fallen comrade. Amundsen and Smith of the Fred Hutchinson Cancer Research Center, writing in the March 21 issue of Cell first set the stage for the story:

    “Faithful repair of broken or damaged DNA occurs by homologous recombination. This process requires a series of enzymes, collectively forming a “recombination machine,” that act on broken DNA. At least three broad classes of activities—helicases, nucleases, and synapsis proteins—constitute parts of this machine and can be provided either by one complex protein or by several separate proteins.”

    They describe two team members, RecBCD and RecF, that act independently under normal conditions. “But recent analysis of an E. coli mutant that lacks RecBCD nuclease activity,” they announce, “normally required for that pathway of recombination, provides a striking example of how functional parts from these two recombination machines can be interchanged.”

    Their minireview entitled, “Interchangeable Parts of the Escherichia coli Recombination Machinery,” also describes how the machines work. They feel this is probably not an isolated example of interchangeable roles: “Perhaps in wild-type cells also, there are situations of altered DNA metabolism not yet recognized in which activities from the two recombination machines interchange to maintain chromosomal integrity.”

    Deep Inside You, Machines Climb Monkey Bars 03/28/2003
    “Within every neuron is a vast protein trail system traversed by a small protein engine called Myosin V,” begins a press release from University of Pennsylvania Health System. But these trails, made of actin, are more like monorails than country paths. For a long time, biophysicists have wondered how myosin V moves along the monorail. How does this little motorcar ride the rail without losing its grip? They know it has two heads that grip the rail, and a tail that holds the cargo. Do the heads (actually more like feet) slide along like an inchworm, or move hand-over-hand? Now at long last, Yale E. Goldman’s team thinks they have solved it. The tiny molecular motors move hand over hand, much like kids in a playground. Goldman explains: “It turns out that myosin tilts as it steps along the actin track – one head attaches to the track and then the molecule rotates allowing the other head to attach – much like a child on a playground crosses the monkey-bars hand-over-hand.” How did they see it? “Using single-molecule fluorescence polarization, we could detect the three-dimensional orientation of myosin V tilting back and forth between two well-defined angles as it teetered along.”

    Molecular Motors: Plants Have Sewing Machines 03/31/2003
    In a discovery that “represents a previously unreported concept and will stimulate further research,” three German biologists have reported that plants have a molecular motor that acts like a sewing machine. Schleiff, Jeilic and Soll of Munich studied an unusually large GTP-binding protein named Toc159 that was previously thought to be just a passive receptor on the surface of the chloroplast. Their analysis shows that “Toc159 acts as a GTP-driven motor in a sewing-machine-like mechanism.”
    They explain that “The translocation of proteins across cellular membranes is a key mechanistic problem for every cell.” Apparently, Toc159 threads its needle by grabbing a precursor protein (preprotein) of the protein needing to get through the membrane. Then, Toc159 empowered by GTP actually pushes the cargo through the Toc75 channel, which expands to accommodate the thread-like protein. Once the cargo is through, Toc159 resumes its position. “Through multiple rounds of preprotein binding and GTP hydrolysis,” the authors explain, “Toc159 will push the polypeptide across the membrane.” Thus it works in a rocking fashion, sending the threads of protein through pores in the cloth of the chloroplast membrane, with two conformational changes and two expenditures of GTP to GDP for each cycle. They suspect other examples of this motor mechanism will be found.
    Source: “A GTP-driven motor moves proteins across the outer envelope of chloroplasts,” in the Proceedings of the National Academy of Sciences online preprints, 3/28/03.

    Stay tuned; cell biologists are continuing to find more molecular machines at work. They’ve already found monorail cars, propellers, motors, fuel cells, electric generators, rheostats, badge readers, shipping and receiving systems, translators, folding equipment, smart bombs, computers, backup tapes, email, and much more. What will be next, a coffee maker?

    Fail-Safe Mechanism Protects Against Gene Re-replication 04/09/2003
    As if you didn’t already have enough to worry about: some 8 million of your cells are dividing at any one time, and they had better get it right, each and every time, because mistakes can be disastrous. During the cell division process (the cell cycle), all those DNA base pairs need to be duplicated so that each daughter cell has a copy. How does the cell guarantee no strand is accidentally copied twice? The cell has a system of checks and balances. A stretch of DNA needs to first obtain a license to be copied. Once the copy is done, the license is removed. Writing in the April 4 issue of Cell, Scottish biologist J. Julian Blow explains how this works:

    “The replication of eukaryotic chromosomal DNA requires the initiation of replication forks from thousands of replication origins. These must be regulated so that none fires more than once in each cell cycle. The cell achieves this by breaking the initiation process into two nonoverlapping phases. In the first phase, occurring in late mitosis and early G1, replication origins are ‘licensed’ for replication by assembly of a prereplicative complex (pre-RC) of initiation proteins. When replication forks are initiated at licensed replication origins during the subsequent S phase, the pre-RC is disassembled, converting the origin to the unlicensed state incapable of supporting further initiation. In order for this system to work properly, the licensing system that assembles new pre-RCs must shut down before S phase starts.”

    He reports on a new function of a multi-talented protein named Ran that is involved in this last step. But it is probably far from the whole story. Blow concludes, “it is unlikely that direct inhibition of licensing by Ran-GTP is the only control. Previous work suggests that several redundant mechanisms might exist to minimize the risk of re-replication occurring, an event with potentially catastrophic consequences.” The Preview article is entitled, “A New Role for Ran in Ensuring Precise Duplication of Chromosomal DNA.”

    DNA Epic Saga a Bigger Production than First Realized 04/12/2003
    “DNA’s Cast of Thousands” is the subject of Elisabeth Pennisi’s commentary in the April 11 issue of Science special issue on “Building on the DNA Revolution.” She recounts the history of the discovery of DNA, and where research is headed. The story line is one of increasing complexity: nucleic acids (1860), a blurry idea of a helical molecule (1951), the genetic code deciphered (1953), then a mushrooming bonanza of discoveries about supporting cast: messenger RNA, transfer RNA, transcription factors, polymerases, repair teams, histones, chromatin, and more. Typical quote: “Again, the process is proving to be even more complicated than researchers initially realized.” Pennisi ends on the recent suggestion that a histone code exists that is “as complex and important as the DNA code.” She ends, “Forty years ago, Brenner and others were convinced that the central questions in molecular biology would be answered well before the turn of the century. Now they know better. The nature of the histone code is just one of many problems whose complexities are left to be unraveled.”

    Traffic Controls in the Cell Prevent Traffic Jams 04/14/2003
    Cells have a variety of cargos that need shipping, including messenger RNA particles, mitochondria, endosomes, lipid droplets, and more. These are continually on the move in the cell, going from one part of the cell to another, where needed. They are carried along by molecular motors that move along tracks called microtubules that have a + (plus) end and a - (minus) end. Each transporter moves toward its specific polarity: kinesin moves toward plus, and dynein moves toward minus. Both motors can grab a piece of cargo simultaneously, but this creates a situation like a boxcar being pulled by engines facing opposite directions. How does the cell coordinate the movements? Is it a tug-o'war, or is there some switching action that coordinates the traffic?
    Apparently the latter. In the April 15 issue of Current Biology, Steven P. Gross of UC Irvine reviews today’s understanding on the subject. Although much remains to be explained, a complex of proteins appears to act like springs to engage or disengage the transporter when necessary, as if putting the idle engine in neutral so the driving engine can have priority. In addition, additional regulation is needed to govern which direction has priority. The result is that even with one-way engines, interference is avoided, so that cargo can move both forward and backward on the track, and even reverse direction if the need arises. By removing these controls, scientists have been able to create traffic jams and pile-ups in a system that otherwise works in smooth coordination.

    Cell Celebrated 04/17/2003
    The April 17 issue of Nature features a collection of reviews on cellular dynamics: cell division, the cytoskeleton, microtubules as molecular machines, molecular motors, and more. In the overview article, Thomas D. Pollard of Yale sees this all as the triumph of the reductionist agenda: i.e., that all this complexity can be explained from simple evolutionary precursors.

    How the Cell Avoids Typos 04/29/2003
    Some of the most intriguing molecules involved in protein manufacture are the set of 20 molecular machines that fasten amino acids onto transfer RNAs. They are called aminoacyl-tRNA synthetases (aaRS) and it is their job to be certain that the correct amino acid is mated to the correct transfer RNA (tRNA). They are like language interpreters, in that they understand both the DNA language of nucleotides and the protein language of amino acids. Just like an interpreter must carefully match an English word to its Chinese equivalent, the aaRS interpreters are key players for ensuring the resulting protein chain is spelled correctly…. in the cell, mistakes can be disastrous, leading to cell death. One difficulty of their job is that some amino acids are very similar to others. Linus Pauling once predicted an error rate of 1 out of 5 (80% accuracy) between isoleucine and valine, since they are differ only in weak van der Waals forces; but experimental evidence shows that the aaRS interpreter scores correctly 2999 times out of 3000 (99.67% accuracy).

    An international team of biochemists publishing in the April 25 issue of Molecular Cell has followed the activity of a couple of these interpreters in unprecedented detail. Before attaching the amino acid, the aaRS machine validates it with a “double-sieve” mechanism, which is like forcing the entrant to open two locks with two independent keys, or making him supply two passwords to two different security guards. It performs both pre- and post-transfer editing. In other words, it validates the incoming amino acid before attachment, and double-checks it after attachment. To begin with, the attachment will not proceed unless the tRNA is charged and the amino acid is activated. The active site for the leucine aaRS machine includes a “discrimination pocket” for the side chain of the amino acid leucine. Simultaneously, it authenticates the adenine of the RNA. If the parts don’t match, or a hacker tries to sneak past, the aaRS machine holds the amino acid in position to be hit by the water-balloon firing squad; an incoming water molecule hydrolyzes both substrates, so that no further harm will come from the mismatched tRNA. The properly-edited tRNA then moves to another machine complex, the ribosome, that joins the amino acids together on an assembly line; here, additional proofreading mechanisms check for accuracy. Then the assembled protein chain moves onto the chaperone for correct folding, then to the intercellular railroad for shipping, etc.
    The team found a critical aspartic acid in the active site of the leucine aaRS interpreter that is “universally conserved” in very different organisms. Mutating it to something else, like alanine, destroys the editing function. So far, scientists have learned about four proteins that can deacylate charged tRNAs, and they have “completely different structural frameworks.” Small changes in these machines also cause a “dramatic effect upon editing.” The accuracy of the aaRS system is just one of many levels of quality control ensuring cell survival. The authors state, “Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.”

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    Orphans in the Genes: An Evolutionary Puzzle Is Growing 01/02/2003
    Now that about 60 microbial genomes have been sequenced, a puzzle that was first brushed off as due to insufficient sampling is refusing to go away, and is getting worse. That’s the conclusion of two geneticists at Ben Gurion University, writing in the Jan. 2 issue of Structure. First, they explain what they mean by orphans:

    “The genomes of most newly sequenced organisms contain a significant fraction of ORFs (open reading frames) that match no other sequence in the databases. We refer to these singleton ORFs as sequence ORFans. Because little can be learned about ORFans by homology, the origin and functions of ORFans remain a mystery. However, in this era of full genome sequencing, it seems that ORFans have been underemphasized.”

    They explain the significance of ORFans to evolutionary theory in a series of unanswered questions :

    “If proteins in different organisms have descended from common ancestral proteins by duplication and adaptive variation, why is it that so many today show no similarity to each other? Why is it that we do not find today any of the necessary “intermediate sequences” that must have given rise to these ORFans? Do most ORFans correspond to rapidly diverging proteins? If so, how rapidly do they diverge, and what are the forces involved in their rapid evolution? Is their rate of change constant or did the rapid changes occur only at specific times? Do these rapidly evolving ORFans correspond to nonessential proteins or to species determinants?”

    It was thought that the numbers of ORFans would drop as more genomes were sequenced, because perhaps more data would provide more matches. With that hope, Siew and Fischer performed a detailed survey of the microbial genomes and graphed the trends. They found that 20% to 30% of sequences fall into the ORFan category. Even though each new genome reclassifies some ORFans as non-ORFans, the number of new ORFans grows faster than solutions. They predict 25,000 ORFans will remain when the 100th genome is sequenced. The authors examine possible explanations for the existence of these sequences of genetic material that are unique to each species. No appeals to sampling error or insufficient data appear to work; the phenomenon is real, and the problem is growing :

    “We conclude that the increasing number of ORFans suggests that our knowledge of nature’s sequence diversity continues to grow, that ORFans may entail an intrinsic phenomenon in evolution, and that a global view of the protein world needs to consider the ORFan sequence families in addition to the large sequence families containing proteins conserved [i.e., unevolved] in numerous organisms.”

    Their goal was not to explain the origin or function of ORFans, but to characterize the extent of the problem. Since each new published sequence is adding more ORFans than finding matches for them within known gene families, “Consequently,” they note, “the total number of ORFans is growing.”

    Duplicate Genes: Fodder for Evolution, or Mechanism for Robustness? 01/02/2003
    Genome sequences reveal a fairly large number of duplicate genes which show varying degrees of sequence similarity to one another. Susumo Ohno suggested 30 years ago that these might provide raw material for evolution; as useless copies, generated by chance during cell division, they might accumulate mutations that could be acted on by natural selection. Now, a paper by Zhenglong Gu et al in the Jan. 2 issue of Nature suggests instead that the duplicates provide robustness for the genome, allowing backup copies that can compensate for a DNA failure. In a News and Views analysis of the paper, Axel Meyer considers this a falsification of Ohno’s hypothesis, and wonders where raw material for evolution can now be found :

    “All of this suggests that gene duplication provides a means of preserving function; even when two copies of a gene have diverged widely, they can still substitute for each other functionally to some degree. This, together with the fact that many genes and gene networks are similar in evolutionarily diverse species, hints that maybe Ohno was wrong after all. Are duplicated genes the stuff of developmental stability and of conservation of function rather than evolutionary innovation? If so, how did the diversity of life around us appear?”

    He suggests some sources, like novel gene-regulatory sequences, but notes that evolutionary theory did not predict this :

    “The discovery of many duplicated genes and parts of genomes has been an unexpected but interesting by-product of genome-sequencing projects. ... We are only now beginning to comprehend just how malleable genomes are, and also how resilient they are in the face of so much genetic perturbation; for instance, rearrangements and duplications of chromosomal segments are also commonplace. Gu et al have provided the first estimate (23-59%) of the contribution of duplicated genes to genetic robustness. This may be one reason why duplicated genes do not diverge to produce pseudogenes, or ‘die’, as quickly or as often as had been predicted on the basis of population-genetics theory.”

    Molecular Rheostats Control Expression of Genes 01/10/2003
    It’s not just the console; it’s the operators, say scientists, that deserve the award for technical excellence. In a Review article in the Jan. 10 issue of Cell, Richard Freiman of Howard Hughes Medical Institute and Robert Tijian of UC Berkeley use adjectives not normally found in dry scientific literature: elaborate, intricate, exquisite, and dramatic. They’re talking not about genes, but the systems that regulate them. A few examples :

    • “The temporal and spatial control of gene expression is one of the most fundamental processes in biology, and we now realize that it encompasses many layers of complexity and intricate mechanisms.
    • ... researchers have identified and partly characterized the elaborate molecular apparatus responsible for executing the control of gene expression.
    • The molecular machinery responsible for controlling transcription by RNA polymerase II (RNA pol II) is considerably more complex than anyone had anticipated.
    • Moreover, working out how subtle changes of the transcriptional machinery can vastly alter activation and repression in the context of the large battery of transcriptional initiation factors will be critical to understanding how elaborate gene expression patterns in metazoan organisms are orchestrated.
    • The finding that posttranslational modification of Met4 by ubiquitin controls selective activation of one set of Met4-responsive genes and not another is remarkable and suggests that cells have evolved [sic] elaborate mechanisms to coordinately control gene expression but, at the same time, discriminate between different pathways by subtle mechanisms we have only begun to appreciate.
    • It is not hard to envision that these lysine residues therefore serve as critical molecular switches that can respond to different signals in highly specific ways. In addition, since most proteins contain many lysine residues, transcription factors may undergo multiple modifications simultaneously or in sequential order, pointing to the possibility of generating complex networks of regulatory events.
    • Clearly, transcription is exquisitely regulated in all organisms ... Future studies in diverse organisms and specialized regulatory pathways should further illuminate how transcription factor modification contributes to the elaborate mechanisms of gene regulation.

    Freiman and Tijian note that gene number cannot be the sole determiner of the difference in outward body types between species, such as between a worm (19K) and a human (30K). There’s much more going on They estimate 10% of the genome is devoted to regulating the expression of genes, and that is largely responsible for the difference between you and the earthworm in your backyard:

    “In other words, the dramatic phenotypic differences between a worm and a mammal can at least partially be rationalized by differences in the complexity of the regulatory code and not merely gene content. ... Regulation by modification not only enhances the functional potential of each individual transcription factor but also provides an effective means of greatly amplifying the functional plasticity of the transcriptional machinery required for combinatorial diversity. This quantum increase in the repertoire of regulatory events ultimately provides the rich tapestry of molecular interactions necessary to direct the diverse arrays of gene expression programs that define complex organisms.”

    The transcription factors they describe in this paper (ubiquitination, sumoylation, acetylation and methylation) are in addition to the recently-recognized “histone code” system (see our November 4 headline about this), and may be even more vital :

    While multiple covalent modifications of histone tails have been well characterized and shown to play a global role in gene expression ..., we postulate that modification of nonhistone regulatory proteins (i.e., transcription factors) will play an equally important and perhaps more specific role in directly modulating transcription.“

    One particularly interesting aspect of their paper is that these regulatory programs, by working synergistically or antagonistically, can provide precision control comparable to a skilled audio technician’s hand on a mixing board: “We propose that potential cascades of modifications serve as molecular rheostats that fine-tune the control of transcription in diverse organisms.” So the regulation of gene expression, not merely gene number or content, may be the main factor that produces a navigating lobster, an archery-champion fish, a sonar-operating bat, or a catapulting horse.

    Cell Contractors Take Delivery On Demand 01/13/2003
    In a Review article in the latest issue of Current Biology, with the Ezekielesque title “Periodic Transcription: A Cycle Within a Cycle,” Linda L. Breeden discusses how cells optimize the time for transcribing genes into proteins. She opens with a picturesque construction analogy :

    “If you were building a house, would it be better to take immediate delivery of every component required to complete the project, or to have things delivered as needed during the assembly process? From the point of view of efficient material management and the accuracy of the assembly process, the latter is the logical choice. Things needed continuously would be kept on hand throughout the process. Things needed only once, especially if they are not easily stored, would be delivered just before they are to be used. With a smaller inventory of things on hand, less time would be required to find things, less breakage would occur and fewer mistakes would be made as a result of mis-identifying parts with similar form but different functions. Given the logic of this strategy, it should be no surprise that it is frequently employed by cells. ... “

    Breeden, who works at the Fred Hutchinson Cancer Research Center in Seattle, discusses recent findings that yeast and bacteria, and probably higher organisms, optimize their gene transcription in remarkable ways. The cell cycle refers to cell division; here is the cycle within the cycle :

    “One remarkable feature shared by all the cell cycle-regulated transcription investigated to date is that each wave of transcription involves transcription factors that are also cell cycle-regulated at the transcript level. ... Some of these cell cycle-regulated transcription factors serve to induce the next wave of cell cycle-regulated transcription. Others serve as feedback regulators to extend, amplify or inhibit another wave of transcription. The result is a continuous cycle of interdependent waves of transcription wherein one wave can affect the timing, composition and/or persistence of an adjacent wave.”

    These strategies serve to control transcript complexity during the cell cycle. Breeden concludes :

    “Logic dictates that reducing the complexity of transcripts at any given time during the cell duplication process would improve its fidelity and efficiency. ... What is clear is that both bacteria and yeast have invested considerable effort into doing just that. The transcriptional circuitry that has evolved [sic] is a series of consecutive and interdependent waves of transcription driven by transcription factors that are themselves cell cycle regulated. It is a simple, yet flexible strategy, with many opportunities for signaling inputs from external sources. Feedback loops have been incorporated which appear to coordinate critical events, and may buffer the cell cycle when conditions change. There are clearly gaps in our understanding, but there is no doubt that this is a general strategy that underlies the yeast and bacterial cell cycles and there is tantalizing evidence that the same may be true in higher cells as well.”

    She leaves it unexplained how these strategies evolved, other than to note that they appear to be conserved from bacteria upwards. “If it’s conserved,” she says, “there’s usually a good reason.”

    Bacterial Cytoskeleton Is a Plastering Artist 01/16/2003
    You learned in school that bacteria don’t have a cytoskeleton. Wrong. Like eukaryotes and all higher organisms, they have internal “cables” but they are not made of actin, but a protein named Mbl. The Oxford team that found this out in 2001 (see Science News) has now found out something even more amazing. Using time-lapse photography and fluorescent dyes, they found that the cables are dynamic structures. They assume a helical shape from one end of the bacterium to the other, and rotate against the inside of the cell wall. Carballido-Lopez and Errington et al, writing in the Jan. 14 issue of Developmental Cell, think they know what they might be doing. They might be plastering layers of material on the inner side of the cell wall. As the cables rotate and the material deposited stretches in the opposite direction, the result is a “multilayered fabric composed of layers of material, each of which is inserted at an angle to the previous overlying layer ... the resultant meshwork structure should be more resistant to shearing forces than a structure in which the stress bearing fibers are inserted in a highly parallel manner.” They are currently testing this model. The fibers, made of peptidoglycan, work their way to the surface and are discarded. The rotating cables inside the organism, therefore, constantly replenish the cell wall from the inside. Cell division is able to operate because the cables are continually remodeled in about 8 minutes on average.

    DNA After 50 Years Continues to Astound Biologists
    01/27/2003
    To celebrate the 50th year of Watson and Crick’s (and Rosalind Franklin’s) discovery of the structure of DNA, the Jan. 23 issue of Nature has a special section entitled, “The Double Helix – 50 Years.” It contains 16 articles by scientists and historians, looking backward and forward, on what we’ve learned so far and what prospects lie ahead.
    In short, DNA is far more complex than the simple double helix we all know from pictures, and there is much we still have to learn. A common theme is that DNA is not a simple, static library, but a very dynamic system, constantly in motion, surrounded by a much more dynamic and complicated set of protein translators, protectors, repairers, and regulators. Words like elegant, exquisite, and marvelous festoon the word parade celebrating “this miraculous molecule” as Helen Pearson calls it in her introductory editorial.
    Philip Ball in “Portrait of a Molecule” describes the incredibly dense packaging process that telescopes 1.8 meters of the DNA ladder into six micrometers of space – a packing ratio of 7,000 to one. Somehow in all the commotion of transcription and cell division, it maintains a “structured chaos” in the nucleus: “It is a constantly changing structure, but not randomly: there is method in there somewhere,” he says, revealing how much remains to be learned. After dazzling the reader with descriptions of the winding, packing, coiling, and supercoiling processes that DNA undergoes in its dizzying dance, he concludes :

    “If all of this destroys the pretty illusion created by the iconic model of Watson and Crick, it surely also opens up a much richer panorama. The fundamental mechanism of information transfer in nucleic acids - complementary base pairing - is so elegant that it risks blinding us to the awesome sophistication of the total process. These molecules do not simply wander up to one another and start talking. They must first be designated for that task, and must then file applications at various higher levels before permission is granted, forming a complex regulatory network .... For those who would like to control these processes, and those who seek to mimic them in artificial systems, the message is that the biological mesoscale [i.e., between molecules and organelles], far from being a regime where order and simplicity descend into unpredictable chaos, has its own structures, logic, rules and regulatory mechanisms. This is the next frontier at which we will unfold the continuing story of how DNA works.”

    Bruce Alberts, President of the National Academy of Sciences, in “DNA replication and recombination”, repeats a theme he has expressed for years, that DNA-protein complexes are best described as interacting molecular machines (a word he uses over a dozen times). He asks how precise these machines have to be :

    “For the first 30 years after Watson and Crick’s discovery, most researchers seemed to hold the view that cell processes could be sloppy. This view was encouraged by knowledge of the tremendous speed of movements at the molecular level ....
    Quite to the contrary, molecular biologists now recognize that evolution has selected for highly ordered systems. Thus, for example, not only are the parts of the replication machinery held together in precise alignments to optimize their mutual interactions, but energy-driven changes in protein conformations are used to generate coordinated movements. This ensures that each of the successive steps in a complex process like DNA replication is closely coordinated with the next one. The result is an assembly that can be viewed as a ‘protein machine’. ... And DNA replication is by no means unique. We now believe that nearly every biological process is catalysed by a set of ten or more spatially positioned, interacting proteins that undergo highly ordered movements in a machine-like assembly.”

    The simple 2D cartoon models of DNA have to go, Alberts concludes; “because most biological subsystems have turned out to be far too complex for their details to be predicted. ... For this reason, we urgently need to rethink the education that we are providing to the next generation of biological scientists.”

    DNA Damage Repair Team Hears Alarm at a Distance 01/30/2003
    DNA, like a ladder, can break, and when both sides break, it’s serious trouble. Cancer and other lethal diseases can result from these double-stranded breaks, or DSBs, which are “the most deadly” of DNA failures. Most of the time, fortunately, there is a response system called ATM that knows just what to do. It can repair both sides of a broken DNA molecule, quickly and efficiently; when not possible, ATM knows how to throw the self-destruct switch to kill the cell so it won’t become cancerous or otherwise dangerous.
    There are many DNA repair mechanisms for many kinds of problems, but most are active during cell division, when there is the highest likelihood for error. ATM, by contrast, works in the resting phase. The heart of the system is a pair of “giant” proteins, normally “locked together in a tight embrace that prevents them from forming any promiscuous liaisons with other proteins” – i.e., their mutual hammerlock keeps them from fraternizing till duty calls. A DSB crisis triggers an alarm; the ATM response separates the repairmen by a process called autophosphorylation, which activates them and puts them to work.
    Christopher Bakkenist and Michael Kastan of St. Jude Children’s Hospital in Memphis, Tennessee, writing in the Jan. 30 issue of Nature, found, to their amazement, that ATM can detect the signal some distance away from the problem. A double-stranded break occurring deep within chromatin-wrapped bundle of DNA can get help fast, even if the repairmen are not near. How does ATM differentiate a real crisis from the normal frenzied activity of cell division, transcription, and translation? Danish cell biologists Bartek and Lukas are amazed at the sensitivity of this emergency response system :

    “Finally, the sensitivity, extent and speed of the ATM response are truly astonishing. Doses of irradiation that cause only a few DSBs in a human cell activate the majority of ATM within minutes. And induction of just two DSBs per cell is enough to induce the crucial ‘autophosphorylation’ of ATM.”

    Much remains to be learned about this paramedic team, but one thing is clear: it keeps us alive. “Our genetic blueprint is constantly assaulted by adverse environmental and cellular influences, such as ultraviolet or ionizing radiation and various chemicals,“ write Bartek and Lukas. “Fortunately, these massive attacks on our DNA are largely counterbalanced by promptly deployed, multifaceted surveillance and rescue operations.”

    Your Electric Personality 02/05/2003
    You shine like a 116-watt light bulb, and run 522 amps of electrical current. To run your power plant, three sextillion protons per second are continually being pumped across enough super-thin membrane, filled with embedded generators, to stretch over three football fields. These and other amazing facts are described by Peter Rich in a Concepts article about mitochondria (our miniature power plants) entitled “Chemiosmotic coupling: The cost of living” in the Feb. 5 issue of Nature.
    He recalls Peter Mitchell’s controversial work in the 1960s that first suggested biological electron transfer was linked to ATP synthesis – a discovery that won him the Nobel Prize. Since then, interest in mitochondria switched on, and increasing knowledge about its electrical activities has lit up the imagination. Here’s part of Peter Rich’s technical description (bracketed notes added):

    “An average human at rest has a power requirement of roughly 100 kilocalories (420 kilojoules) per hour, which is equivalent to a power requirement of 116 watts - slightly more than that of a standard household lightbulb. But, from a biochemical point of view, this requirement places a staggering power demand on our mitochondria. Mitchell’s work showed that the electrochemical gradient of protons across the inner mitochondrial membrane that drives ATP synthesis is roughly 200 mV, and most of this is the electric field component.

    If it is assumed that 90% of human power is provided by the protons that are transferred through the ATP synthase, then the transmembrane proton flux would have to represent a current of 522 amps, or roughly 3 x 10exp21 protons per second. ... Assuming a conversion efficiency that is close to unity [i.e., 100% efficiency], ATP is reformed at a rate of around 9 x 1020 molecules per second, equivalent to a turnover rate of ATP of 65 kg [143 lb.] per day and with much higher rates than this during periods of activity. This output is itself powered by the oxygen-consuming respiratory chain.

    A typical adult male consumes around 380 litres of oxygen each day, and top athletes can sustain rates that are ten times greater for limited periods. Most (90%) of this oxygen is reduced to water by the terminal respiratory-chain enzyme, cytochrome oxidase. The inner mitochondrial membrane contains around 0.4 nanomoles of this enzyme per milligram of protein. It can work at a rate in excess of 300 electrons every second, but probably operates at an average rate of no more than 50 per second. Hence, an average human will need 2 x 10exp19 molecules [20 quintillion] of cytochrome oxidase to support oxygen consumption. With the inner mitochondrial membrane having a lipid/protein weight ratio of 1:1, the cytochrome oxidase would be associated with about 70 ml of lipoprotein membrane. However, the membrane’s thickness - only 6 nm [6 billionths of an inch] - means that the surface area of the inner mitochondrial membrane in an average human would be around 14,000 m2.”

    Rich, biologist at University College, London, is focused not so much on the wonders of the system but the “cost of living” – the “herculean task” that eukaryotes undergo to synthesize ATP for energy consumption, through a membrane that acts as a capacitor, and ATP synthase rotary motors that take the protons and make ATP from ADP and phosphate. “The energy thus stored,” he explains, “can be released by ATP hydrolysis, a reaction that is used by the myriad energy-requiring enzymes that maintain cellular function.” He points out that mutations or defects in the mitochondrial DNA are implicated in physiological disorders, and probably increase throughout our lifetimes. So as we age, our light bulbs eventually burn out.

    Scientists Pump the Flagellum Engine 02/10/2003
    Japanese researchers have found that flagella, the whiplike propellers that make bacteria swim, can get flooded with too many protons if the pH is lowered inside, reports Nature Science Update. Like a flooded car engine, the motors come to a stop. But they can run fine again if the artificially-induced pH change is reversed. The article concludes by discussing the functional specifications of these molecular machines:

    “This is a motor with quite remarkable properties,” says Robert Macnab of Yale University in New Haven, Connecticut, who studies the assembly of bacterial motors. “It runs like a battery, moves like a ship’s propeller, has a gear switch so it can rotate in either direction, and it’s under the control of information from [the] environment. These are biological functions at their most simplified form, and yet there are 60 different types of components in this little engine.”

    Kendall Powell explains the interest in these motors: “Researchers are keen to understand such chemically driven biological motors, which are only millionths of a millimetre across, as electronics do not work on this scale.”

    Protein Machine Does Gymnastics 02/13/2003
    Scientists are bringing into sharper focus an amazing molecular motor named dynein. Dynein is responsible for much of the movement in the cell: the whiplike action of sperm tails, the sweeping action of cilia, and the ferrying of cargo down the microtubule intracellular railroad. The UK research team of Stan Burgess et al in the Feb. 13 issue of Nature imaged thousands of the little molecules (large by protein standards, with a molecular mass of over 500,000) that work something like railroad handcars. They have a ring-shaped hexagonal head of six AAA proteins to which is added a C-terminal domain. Emerging out of one side and in the same plane as the ring is a stalk, which has a structure on the end that attaches to the microtubule. Emerging out the other end is a stem that attaches to whatever cargo needs to be transported. The stem is fastened to the ring by a linker, that seems to act like a ratchet on a gear during the cycle.
    How does it work? Though the details are still fuzzy, it appears that ATP hydrolysis occurs in the central ring, or head domain; i.e., energy is extracted from ATP, producing ADP and phosphate, putting the machine into a “cocked” state. This causes a conformational change (parts moving in relation to one another) resulting in a 34o rotation of the ring relative to the linker. The head domain rolls in relation to the stem, producing mechanical spring energy. Since the stalk and stem have some flexibility, they are “capable of storing elastic strain energy when the molecule develops force against a load.” The movement pops out the ADP, and then the mechanism springs back to its cocked position; the so-called “power stroke.” Simultaneously, another ATP energy pellet enters the engine for the next cycle.
    The angle between the stalk and stem thus changes back and forth in a rocking fashion, producing mechanical leverage, as the linker continually engages and disengages in the central ring, like a hook catch on a gear. As a result, the dynein motor slides down the microtubule monorail in 15-nanometer jumps. But that’s not all; there is two-way communication between the tip of the stalk and the engine in the head, and even more amazing regulatory mechanisms that tell the motor where and how fast to go.
    In their News and Views write-up on the paper, entitled “Molecular motors: A magnificent machine,” Richard B. Vallee and Peter Höök consider this a remarkable gymnastic ability that is rarely seen in motor proteins. The dynein machines actually use the chemical energy stored in ATP to produce force and carry out work. They point out that this action occurs many times per second in the molecular motor.
    If you can’t reach the Nature article, the BBC News has a summary of it that likens dynein to engines with pistons that make wheels turn. One of the researchers is quoted likening the system to a railway network: “Our body is full of proteins which form tracks. Along these tracks, molecular motors are the locomotives, transporting a variety of cargoes to wherever they are needed”

    Cell Nucleus More Than Just a Bag of Chromosomes 02/19/2003
    Scientists at Johns Hopkins Medical Institutions are finding that the nucleus of the cell is not just a passive storage area for genetic information. Kathy Wilson told the AAAS meeting on the 17th that the nucleus is “is really the cell’s mothership, a crucial and very active source of information, support and control.” One amazing feat occurs during cell division. Chromosomes are pulled apart outside of the nucleus, so the nucleus must disappear during the process. It does not just fall apart. Wilson described it as “an orchestrated process similar to the pulling apart of the chromosomes. It seems to involve the same structures and the same tiny motors. It’s almost a practice run for moving the chromosomes”

    Cell Repairs its RNA, Too 02/20/2003
    The cell has elaborate ways to safeguard its genetic library by repairing DNA, but now scientists are finding the same enzymes can also repair RNA. In the Feb. 20 issue of Nature, Begley and Samson of MIT discuss the findings of Aas et al that RNA methylation damage can be repaired by the same AlkB enzyme that repairs DNA. This is surprising because RNA and proteins were considered more expendable than DNA, but they explain why it makes sense :

    “Why, though, should it be necessary to repair damaged RNA? The answer could be that although DNA is the final arbiter of genetic information, RNA is essential for the most basic biological processes. RNA-based primer sequences are required for DNA replication; and mRNAs, transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are all needed during the elaborate process of protein synthesis. Even the formation of peptide bonds by ribosomes (the cell’s protein-making machines) turns out to require catalysis mediated by rRNAs. Moreover, a battery of small, non-protein-coding RNAs regulates a variety of other cellular processes.”

    So maintaining RNA integrity is important for proper cellular function. And repairing damaged RNA may be more efficient than destroying it and starting again. Ribosome assembly is a complex, energy-intensive process, and it is not hard to imagine that the thrifty repair of damaged rRNA would be preferable to disassembling or discarding an entire ribosomal particle.

    Another surprise is that the repair mechanism seems to be able to distinguish between DNA and RNA, and between toxic methylation damage and normal biological methyl groups attached to some RNAs. Begley and Samson think it not unlikely that DNA and RNA might overlap in other ways, such as in cell signalling. Update 06/16/2003: In the June 17 issue of Current Biology, Alfonso Bellacosa and Eric G. Moss from the Fox Chase Cancer Center in Philadelphia remind us that “RNA in a cell is subject to many of the same insults as DNA“ and that “the ‘information content’ of cellular RNA is greater than that of the chromosomal DNA” because almost all of RNA’s sequences have functional significance (messenger RNA and transfer RNA), whereas only 3% of the DNA has coding potential. Since RNA shows significant response to anticancer agents, the authors suppose that newly-discovered RNA repair pathways are important for preventing cancer:

    “A cell has a great investment in its RNAs – they are working copies of its genomic information. The study of mRNA biogenesis in the last few years has revealed an elaborate surveillance mechanism involving factors such as the UPF proteins that culls aberrantly spliced mRNAs and mRNAs with premature termination codons. There might be a hint that such RNA quality control mechanisms go awry in cancers, just as DNA quality control mechanisms do, where aberrantly spliced transcripts accumulate in a tumor. Now that the gates are open, we may have a flood of studies on the RNome [the RNA genome] stability and cancer.”

    Another Rotary Motor Found in Cells 02/24/2003
    Another member of the ATPase (ATP synthase) superfamily has been shown to rotate and produce three ATP per cycle. The well-known FoF1-ATP synthase was imaged in rotation about five years ago. Another enzyme, VoV1-ATPase, was known to be structurally similar and has been assumed to rotate also, but experimental evidence was lacking. The Japanese have done it again. They attached a bead to the stalk and imaged the tiny molecular machine rotating counterclockwise at about 144 rpm, which they assume is the natural rotation rate without the bead attached.
    VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis in Archaea and some eubacteria. FoF1-ATP synthase resides in the mitochondria and chloroplasts; VoV1-ATPase is embedded in various intracellular acidic compartments. This enzyme’s D subunit acts like a rotor shaft, analogous to the gamma subunit of F1ATPase. The experimental results are written up in the Proceedings of the National Academy of Sciences online preprints for Feb. 21.
    How they work: The Fo and Vo subunits of the machines are embedded in the membranes and use proton motive force to rotate. The F1 and V1 subunits are where ATP synthesis takes place. They contain six lobes that are acted on by a rotor shaft, or camshaft, attached to the rotating portion. The six lobes come in pairs. As the camshaft turns, it causes each pair to cycle through the manufacturing steps: load the ingredients (ADP and phosphate), squeeze them together into ATP, then eject the ATP into the surrounding medium. Each pair is undergoing one of these stages every 120o turn of the camshaft, so that 3 ATP are produced for every full turn. ATP is the energy currency used by most processes in the cell. On a busy day, your miniature motors can recycle an amount of ATP equal to or exceeding your body weight.

    Your Model Train Set 02/25/2003
    Model train enthusiasts never had it so good. Imagine five different models of finely-crafted engines, all in perfect working order, and enough track to cover a city. That’s what each of us has, right now, inside our cells. But don’t feel top dog; even lowly bacteria have them, too. To prove we’re not making this up, read “The Molecular Motor Toolbox,” a Review article in the current issue of the journal Cell, by Ronald D. Vale of the Howard Hughes Medical Institute. He begins:

    “A cell, like a metropolitan city, must organize its bustling community of macromolecules. Setting meeting points and establishing the timing of transactions are of fundamental importance for cell behavior. The high degree of spatial/temporal organization of molecules and organelles within cells is made possible by protein machines that transport components to various destinations within the cytoplasm.”

    Vale reviews the five major motor engine families that ferry cargo around the cell: actin, dynein, conventional homodimeric kinesin, heterotrimeric kinesin II, and Unc104/KIF1. These engines show remarkable flexibility and diversity in living things, from plants to sea squirts to fungi to worms, and are highly conserved from the smallest organisms to the largest. What about the switching? What keeps the engines from colliding on the tracks?

    “To achieve law and order on the intracellular highways, the multiple cargo-carrying motors in a single cell must be regulated. In the majority of animal cells, individual organelles switch frequently between anterograde (microtubule plus-end-directed) and retrograde (minus-end-directed) movement .... In most cells, relatively little is known about the regulation and coordination of bidirectional motion. ... individual cargoes move primarily unidirectionally in these extended processes, and a switch in direction occurs when cargoes reach the ends of these elongated structures.”

    There is an unknown switching mechanism at so-called “turnaround zones” on the microtubules that dynein and kinesin engines travel on.

    “The microscopic observations of cargo transport in axons and flagella raise a number of similar questions. How do the opposite polarity motors, kinesin and dynein, coordinate their activities? What kind of machinery processes the incoming cargo and switches motor direction at the ‘turnaround’ zones? Molecular answers to these questions are beginning to emerge but are far from complete.”

    As a sidelight, another review article in the same issue of Cell by a team from UC San Diego describes how these motors are involved in tugging the chromosomes apart during cell division (mitosis). In fact, the whole Feb. 21 issue is a good source for current knowledge about the cell’s inner workings: mitochondria, cell division, signalling, transport, etc. But back to our story.
    Vale points to fascinating indications that the motors signal each other and coordinate their actions. After discussing some of these possibilities, he concludes, “Fifteen years ago, only a few molecular motors were known. In contrast, complete inventories of molecular motors are now available in a number of diverse organisms. While these remarkable accomplishments have answered many questions, the genomic inventories also have exposed many areas of ignorance.” Well, back to the lab; gotta get to “work.” Biochemistry can be fun. You get to play with miniature railroads.
    Nature Science Update reports that NASA engineers are studying the intracellular railroad for spacecraft ideas. UCLA got a $30 million NASA grant to begin the Institute for Cell Mimetic Space Exploration, whose mission is to “come up with biology-inspired devices that could facilitate space travel 30 years from now.” Some of the plans include imitating actin.

    Footnote: In the same issue of Cell, an Austrian team discusses the state of knowledge about meiosis (cell division for sexual reproduction). They note that there is no evidence for evolution of this highly complex series of processes :

    “In summary, the behavior of chromosomes in meiosis is much more complex than in mitosis. Additional demands such as chiasmata formation, mono-orientation of sister kinetochores, protection of centromeric cohesion, and prevention of DNA replication between the two divisions are imposed upon the chromosome segregation machinery. These processes are discussed in detail in the following sections. Despite its greater complexity, there is no clear evidence that meiosis evolved later than mitosis. There are, for example, no extant lineages that appear to have split off the eukaryotic tree before the evolution of meiosis (Cavalier-Smith, 2002).”

    Footnote 2: Another molecular motor story appeared on EurekAlert Feb 25. Stanford scientists are studying kinesin, the “workhorse of the cell,“ which hauls chromosomes, neurotransmitters and other vital cargo. Joshua Shaevitz describes it: “This is one of the most efficient engines anyone has ever seen. Some estimates put it at near 100 percent efficiency. It’s an amazing little thing.” His colleague Charles Asbury chimes in with elegant prose, “Kinesin is an example where Mother Nature kicks our butt. For me, I’m motivated just by understanding how this fascinating thing works.”

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    Two For the Price of One: Did Transfer RNA Arise From Complementary Genes? 11/01/2002
    The latest (Oct 27) issue of Molecular Cell has a novel theory on how transfer RNA evolved. In a letter to the editor, Charles Carter and William Duax revive the 1995 Rodin-Ohno hypothesis that suggests complementary strands of DNA can code for different proteins. To understand this, we need to back up and learn a little about transfer RNA. DNA translation, you may recall, starts when enzymes unwind a strand of DNA to expose a gene, and a messenger RNA molecule forms by base-pairing with the exposed DNA nucleotides (the “letters” A, C, T, and G). The resulting messenger RNA molecule (mRNA), like a long computer tape bearing the code for a protein, exits the nucleus and approaches a ribosome. There, individual transfer RNA molecules (tRNA), each carrying a specific amino acid on one end and a three-letter RNA codon on the other, mate with specific parts of the mRNA by base-pairing, and the amino acids on the other end join together into a protein chain. This much is elementary biochemistry, illustrated beautifully with computer animation in the film Unlocking the Mystery of Life. The process is actually much more complicated, requiring a host of helper enzymes at every step.

    One set of helpers that is extremely critical to the accuracy of the operation comprises the aminoacyl-tRNA synthetase family (aaRS). These are the enzymes that join the appropriate amino acids to the transfer RNA molecules. There are 20 of these aaRS enzymes, one for each of the 20 types of amino acids used in proteins. What is most interesting and amazing about them is explained by James F. Coppedge in his book Evolution: Possible or Impossible?:

    “There seems to be no natural attraction between an amino acid and its own transfer-RNA, so something must bring them together. It is as if there were two languages, and neither party understands the other except when there is an interpreter to bridge the gap. This essential task is done by a special group of enzymes which match the different tRNA’s and amino acids. One part of each such enzyme fits just its own particular kind of amino acid and no other. Another part of the enzyme interacts with its own type of tRNA. In plain language, it can be pictured as follows: the enzyme grasps its amino acid and its tRNA and fastens them together. (p. 147)

    In the Molecular Cell article, Carter and Duax recognize the challenge that this complex arrangement presents to evolutionary theory :

    “The fidelity of protein synthesis resides almost entirely in the 20 aminoacyl-tRNA synthetases (aaRS), which acylate their cognate tRNAs with the appropriate amino acid. The origin of codon-dependent translation presents a challenging intellectual problem in biology, owing to the apparently irreducible complexity represented by their simultaneous appearance. Key to the conundrum is that contemporary aaRS divide into two classes each with ten enzymes (Eriani et al., 1990 ), whose respective architectures have quite unrelated homologies (Cusack et al., 1990 ). Moreover, catalytic domains in class I and II aaRS:tRNA complexes from corresponding subclasses have complementary shapes that recognize nonoverlapping surfaces on the tRNA acceptor stems (Ribas de Poublana and Schimmel, 2001 ). Pairwise binding between classes may therefore have protected tRNAs early in evolution, and the class division likely dates from the dawn of biology.”

    Drawing from an analogy with a gene found in a freshwater mold that seems to code for two different proteins, depending on whether the primary strand of the gene or its complementary strand is translated, Carter and Duax propose that genetic complementarity is behind the origin of the two classes of aaRS enzymes: one class evolved off the primary “sense” strand of DNA, and another class evolved off the complementary “antisense” strand. From the original primordial split, the 20 aaRS enzymes arose: “Two synthetases, coding from that repertoire, might similarly have sufficed to produce recognizable protein folds. Subclass speciation via gene duplication then would have enriched the coding repertoire.”

    Cell Memory “Borders on the Miraculous” 11/04/2002
    Just when you thought the DNA code was mind-boggling enough, along comes the histone code. Another coding system somehow helps the cell remember itself: whether it is a blood cell, or a nerve cell, or a muscle cell. While all these cells in your body have the same genetic code, some kind of epigenetic (above-gene) code is telling it what genes need to be turned on. The Nov. 1 edition of the journal Cell has a review by Bryan M. Turner of the University of Birmingham (UK) called “Cellular memory and the histone code” that waxes enthusiastic about this cutting-edge mystery :

    “It is an obvious but easily forgotten truth that cells must have a mechanism for remembering who they are. A cell’s identity is defined by its characteristic pattern of gene expression and silencing, so remembering who it is consists of maintaining that pattern of gene expression through the traumas of DNA replication, chromatin assembly, and the radical DNA repackaging that accompanies mitosis [cell division]. The mechanisms by which around 2 m [two meters, about 6 feet] of DNA is packaged into the cell nucleus while remaining functional border on the miraculous and are still poorly understood. However, we do know more about the first stage in this packaging process, the nucleosome core particle. This structure comprises an octamer of core histones (two each of H2A, H2B, H3, and H4), around which are wrapped 146 base pairs of DNA in 1 3/4 superhelical turns (Luger et al., 1997 ). The reduction in DNA length produced by this histone-induced supercoiling is modest, but is an essential first step in the formation of higher-order chromatin structures. In recent years it has become clear that the nucleosome has an additional role, perhaps equally important and conserved, namely regulation of gene expression. Particularly exciting is the growing probability that the nucleosome can transmit epigenetic information from one cell generation to the next and has the potential to act, in effect, as the cell’s memory bank.”

    Turner describes how the histones have tails that are exposed on the exterior of the nucleosome. It is on these tails where a variety of enzymes can rearrange some of the amino acids, providing a “rich source of epigenetic information.” So how is the code maintained and translated?

    “It has been suggested that specific tail modifications, or combinations thereof, constitute a code that defines actual or potential transcriptional states (Jenuwein and Allis, 2001; Richards and Elgin, 2002; Spotswood and Turner, 2002). The code is set by histone modifying enzymes of defined specificity and read by nonhistone proteins that bind in a modification-sensitive manner. In order to realize its full information carrying potential, the code must use combinations of modifications. This requires not only proteins that can read such combined modifications, but mechanisms by which they can be put in place and maintained. Recent papers have provided new insights into how specific combinations of tail modification might be generated and revealed mechanisms by which the modification of one residue can determine that of another.

    Turner discusses in some detail the types of reactions already known and puzzles that remain to be solved. The histone code appears quite different from the DNA sequence of letters; it is more a sequence of events: “Viewed in this light, the histone code can be seen as part of a sequence of events, possibly involving structural and catalytic proteins and RNAs, whose end result is a functionally stable chromatin state.” At times in the article the complexity of all of this seems to get to him; “To add further complexity,” begins one sentence. Near the end, Turner remarks wryly, “It is in the nature of scientific progress that simple ideas, like people, grow more complex with age.”

    Defective Proofreading Causes Cancer 11/12/2002
    Scientists at University of Utah School of Medicine mutated genes in mice responsible for proofreading DNA, and saw 94% of them get cancer. Writing in the Nov 12 online preprints of the Proceedings of the National Academy of Sciences, they stated, “Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3'-5' exonucleolytic proofreading, mismatch correction, and DNA damage repair.” They induced a point mutation in DNA polymerase delta, one of the molecular machines with a proofreading domain, and the high incidence of tumors resulted. Only 3-4% of the mice without the mutation developed cancers.

    DNA Translator Does the Twist 11/16/2002
    A molecular protein machine responsible for translating DNA in a “primitive” cell does some pretty amazing gymnastics, scientists have discovered. Writing an extended research paper in the Nov 15 issue of Science, two biochemists from the Howard Hughes Medical Institute (Yim and Steitz) found that the RNA Polymerase (RNAP) of T7 bacteriophage is quite the contortionist. Lacking the larger genome of eukaryotes, its DNA translation equipment has to get by with less, so it performs three large conformational changes on one end, and additional shifts on the other: “The transition from an initiation to an elongation complex is accompanied by a major refolding of the amino-terminal 300 residues. This results in loss of the promoter binding site, facilitating promoter clearance, and creates a tunnel that surrounds the RNA transcript after it peels off a seven-base pair heteroduplex.” This involves seven subunits rotating 140 degrees and shifting 30 angstroms, then one subunit stretching out over twice its initial length. Then comes the grand finale:

    “Perhaps the most unprecedented conformational change involves residues 160 to 190, which not only extensively refold, but move about 70 Å from one side of the polymerase to the other. This region refolds from a short helix and an extended loop into a pair of antiparallel helices (H1 and H2/3). The newly formed compact structure, named subdomain H, forms part of the RNA-transcript exit tunnel and contacts the 5' end of the RNA transcript on one surface and the nontemplate DNA on the opposite surface.”

    The other end also undergoes shape-changes to create an exit tunnel for the RNA copy of the DNA. This “massive structural reorganization” of the protein machine causes it to form a protective tunnel, positively charged on the interior, in which the delicate work of translation can occur accurately. The tunnel interior melts the DNA into two strands, shunts the non-coding strand safely to the side, brings the RNA copy elements in and binds them to the DNA template. As the machine progresses down the track, it twists and bends the DNA against its natural inclination. This then supplies the energy to open up the strands and create a “transcription bubble” where the RNA letters (nucleotides) are mated with the DNA code in the “active site”. The tunnel has just the right shape to allow the RNA elements to come in. RNAP first has to attach to the DNA at a specific starting point called a promoter; this is the “initiation” phase. It appears that another protein called lysozyme regulates RNAP by binding to it, and preventing it from entering the “elongation phase” where all the gymnastics occur prior to the real translation work. In the initiation configuration, RNAP can produce only short chains (oligonucleotides) of RNA. The authors puzzle over whether there is a reason for this:

    “One might ask why the abortive synthesis of short oligonucleotides exists and why the enzyme might not be “designed” to carry out the stable RNA synthesis that occurs in the elongation phase right from the start. The initiation of RNA synthesis at a particular site that is required for specific gene expression and regulation as well as the need for de novo, unprimed synthesis necessitates binding of the polymerase at a specific DNA location, the promoter. Furthermore, the binding of T7 RNAP to both the promoter and the downstream DNA appears to be essential for opening the bubble. Because short transcripts (2 to 4 nt) cannot form stable heteroduplexes, polymerase leaving the promoter prematurely would presumably lead to bubble closure and transcript displacement by the nontemplate strand. An enzyme locked in the elongation mode conformation seems unlikely to be capable of specific initiation and bubble opening. “

    The authors also found that point mutations in certain spots either broke the machine or made it translate much less efficiently. Eukaryotes have additional protein parts in their translation machinery that do not require the contortions done by RNA Polymerase in these ultra-miniature life-forms.

    Bacteria Borrowed Each Other’s Photosynthesis Technology 11/22/2002
    How could bacteria evolve the complex processes of photosynthesis five times separately? By technology sharing. That’s a new idea propounded by a team at Dalhousie University in Nova Scotia, reports Elisabeth Pennisi in the Nov. 22 issue of Science. They didn’t have to invent it from scratch each time; they got the parts at the swap meet, via a process of lateral gene transfer.

    Is Darwin’s Tree of Life Visible in the Genes? 11/26/2002
    Two papers in the Proceedings of the National Academy of Sciences online preprints (11/25) complicate the task of deducing common ancestry from genetic codes. One by Kerry L. Shaw of University of Maryland is entitled, “Conflict between nuclear and mitochondrial DNA phylogenies of a recent species radiation: What mtDNA reveals and conceals about modes of speciation in Hawaiian crickets.” She concludes from her comparison of phylogenies built from nuclear and mitochondrial DNA that “speciation histories based on mtDNA alone can be extensively misleading.”

    Another paper by three Penn State geneticists is called, “Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics.” They investigated the technique of Bayesian inference, a popular method for inferring causation, and found it too “liberal” compared to the more “conservative” bootstrap method. They write, “Bayesian analysis can be excessively liberal when concatenated gene sequences are used, whereas bootstrap probabilities in neighbor-joining and maximum likelihood analyses are generally slightly conservative. These results indicate that bootstrap probabilities are more suitable for assessing the reliability of phylogenetic trees than posterior probabilities and that the mammalian and plant phylogenies may not have been fully resolved.”

    New Origin of Life Theory — Sans Primordial Soup – Turns Traditional View Upside Down 12/04/2002
    In a press release, the Royal Society proclaimed a revolutionary new theory for the origin of life that “is set to cause a storm in the science world and has implications for the existence of life on other planets.” According to William Martin and Michael Russell, life did not begin in a warm little pond or primordial soup, but was incubated in iron sulfide rocks at the bottom of the sea. They believe this improves the odds that life will be found on other planets. Their hypothesis is to be published in the Jan. 2003 issue of Philosophical Transactions - Biological Sciences.

    In the Dec. 2002 issue of the Royal Society’s Biological Proceedings, Krakauer and Sasaki introduce an unusual speculation about the origin of life. They argue, surprisingly, that randomness actually helped life develop. In their abstract, they have turned all the usual drawbacks into benefits :

    “The origin of stable self-replicating molecules represents a fundamental obstacle to the origin of life. The low fidelity of primordial replicators places restrictions on the quantity of information encoded in a primitive nucleic acid alphabet. Further difficulties for the origin of life are the role of drift in small primordial populations, reducing the rate of fixation of superior replicators, and the hostile conditions increasing developmental noise. Thus, mutation, noise and drift are three different stochastic effects that are assumed to make the evolution of life improbable. Here we show, to the contrary, how noise present in hostile early environments can increase the probability of faithful replication, by amplifying selection in finite populations. Noise has negative consequences in infinite populations, whereas in finite populations, we observe a synergistic interaction among noise sources. Hence, two factors formerly considered inimical to the origin of life - developmental noise and drift in small populations - can in combination give rise to conditions favourable to robust replication.”

    Molecular Motors — “Remarkable Machines” 12/17/2002
    In a Commentary in the 12/16 online preprints of the Proceedings of the National Academy of Sciences, John Murray of University of Pennsylvania School of Medicine reviews what is known about molecular motors in the cell, particularly the linear motors (like trains on a track), which for all intents and purposes are true motors that actually move things and perform work that is measurable. Linear motors move along tracks (actin filaments or microtubules) in discrete steps of predetermined length (8nm for kinesin), and are polarized to move in only one direction; yet there are so many pairs of tracks and cars that the cell has no problem getting cargo to any point desired. Murray is especially intrigued by how motors like myosin and kinesin combine chemical process with mechanical actions in cycles that involve feedback between them; how do they do it?

    “The details are still actively sought, but the overall process goes like this. One or more of the chemical transitions in the catalytic center causes a specific small movement of neighboring parts of the protein, and this small movement is mechanically amplified into a much larger movement of the core motor domain relative to the “tail.” Some of the chemical transitions also dramatically change the affinity of the head for the track. Conversely, binding of the head to the track alters the rates of some specific chemical transitions. Glossing over all of the uncertain bits, the net result (see Fig. 2) is that the head swivels its way along the track while ATP is hydrolyzed (i.e., in the entertaining but anatomically bizarre terminology of this field, the passive tail is pulled along by a wagging head)”
    .
    Murray also finds it intriguing that many of the motors have two head domains. The two heads talk to each other constantly; neither can produce the motion by itself. In the case of kinesin, scientists are still trying to figure out whether the heads “walk” in a hand-over-hand fashion or move instead like an inchworm. He describes how “some motors work in groups, organized in ordered arrays of motors and tracks such as the interdigitating myosin and actin filaments of muscle tissue.” Yet other molecular motors work alone, shuttling their cargo on single tracks like handcars on a monorail.
    The thrust of Murray’s commentary is hope that a new technique might help sort out the interaction of the two heads so scientists can discover how they work. In passing, he notes some of the performance specifications of these motors:

    “In addition to processivity, other experimentally accessible parameters of a motor include its maximum velocity (~800 nm/s for kinesin; 5-50,000 nm/s for other motors), maximum force (~6 pN for kinesin; step size X force is limited by the energy of ATP hydrolysis, ~100 pN/nm per molecule, roughly 25 kT), maximum rate of ATP hydrolysis (~20/s per head for kinesin; 0.5-100/s for other motors), and affinity for tracks.”

    By affinity for tracks, he means that these motors are attracted to the surface of the microtubules on which they zip around by forces of chemistry; like a wall climber with magnetic shoes, a kinesin can go 50-250 steps without falling off. The velocity measurements mean, in plain English, that these little motors are speed demons. Using his numbers and assuming a body length of 8nm for kinesin, if translated up to race car size, it would go 100 body-lengths per second; for a 12-foot race car, that’s over 800 mph. For the 50,000 nm/s motor, could it really be ... 400,000 mph?

    Cell Chaperones: Did Generalists Evolve From Specialists? 12/30/2002
    “Chaperones” are barrel-shaped protein machines in the cell whose task is to provide a safe folding place for newly-assembled polypeptides. One of their remarkable properties is the ability to help fold a wide range of proteins, something like a car wash that fits all models. Instead of the cell needing to maintain a specialist for each protein, a generalist does the job for most. In the Dec. 27 issue of Cell, researchers at Howard Hughes Medical Institute wrote up their experiment on “Directed Evolution of Substrate-Optimized GroEL/S Chaperonins.” They took a chaperone named GroEL/S and “evolved” it to do a better job at folding one protein named GFS, but found that as it got better at being a specialist, it got worse at being a generalist:

    “These findings reveal a surprising plasticity of GroEL/S, which can be exploited to aid folding of recombinant proteins. Our studies also reveal a conflict between specialization and generalization of chaperonins as increased GFP folding comes at the expense of the ability of GroEL/S to fold its natural substrates.”

    They feel this might help explain the evolution of these general-purpose folding stations: “Our results establish that the structure and reaction cycle of GroEL/S give it great plasticity, allowing the chaperonin to be tailored to increase the efficacy of folding of particular substrates.” Their champion GFP-folding specialist, however, lost in the all-around: “GFP-optimized chaperonins often led to significant growth defects.” Eukaryotes have a combination of generalist and specialist chaperones. The authors feel the conflict between efficiency and adaptability drives the evolution of these molecular machines. The authors note that proteasomes and nuclear pores are also generalists, but achieve their skill differently; for those structures, specialized “adapter proteins” bind to the substrate and then to the complex, something like tow bars specific to trucks, tractors, sedans and motorcycles first mating to their specific vehicle, allowing them to be all hooked to a common conveyor belt.

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    Do Enzymes Evolve From Nonenzymes? 10/02/2002
    A paper in the Oct. 2 issue of Structure compared enzymes with nonenzyme homologs to see if either had evolved into the other. In “Sequence and Structural Differences between Enzyme and Nonenzyme Homologs,” three UK women biochemists have no doubts that evolution has shaped enzymes into their repertoire of functions: “Ancestral genes have been duplicated, mutated, and combined through evolution to generate the multitude of functions necessary for life.” Yet deep in the paper, it is clear that most of their findings show that most homologs appear to have lost enzymatic function: “The examples presented suggest that the evolution of a nonenzyme from a catalytic precursor is more common than the reverse scenario, that is, the design of a catalytic function on an ancient nonenzyme domain.” They found 12 examples of enzymes losing catalytic function, and five of nonenzymes gaining it; but “In all five ‘nonenzyme to enzyme’ examples ... nature appears to have exploited the specific binding properties of the catalytically inactive precursor.”

    Your Immune System: How the Assassins Recognize the Terrorists 10/03/2002
    Killer T cells, like roving assassins in a search and destroy mission, look for viral terrorists and obliterate them in their hideouts. But first they have to recognize who is friend or foe, and in auto-immune diseases or tissue rejection, sometimes mistakes are made. How the cell flags its contents, and how the T cell detects it and responds, is a complex and mysterious process. Scientists are nearing completion of understanding the general picture from start to finish. One question that remained was how a system that is highly conserved from mice to men could produce a unique “scent” at the cell surface, so individual that your immune system can sense the difference between “you” and “foreign.” An important piece of the puzzle has recently been identified and reported in the Oct 3 Nature by U.C. Berkeley biochemists. But to understand it, we have to back up and create some word pictures, or else get bogged down in abstruse jargon. Pardon us in advance for the silly analogies and mixed metaphors; the reality is really quite amazing.

    Killer T cells recognize body cells infected with a virus because each cell has a sophisticated system of wearing its innards on the outside. An individual cell can have about 10,000 flags on its surface, composed of pieces of every protein found in the interior. These flags, like sausages always nine amino-acid links long, are mounted on flagpoles, or rather meatpoles, called MHCs. How do they get there? Well, as proteins inside outlive their usefulness and are tagged for recycling, a barrel-shaped meat cleaver called a proteasome chops them into sausages up to 15 units long. Sent back into the cell, most are quickly seized upon by roving dogs (aminopeptidases), but some manage to make it into the subway (endoplasmic reticulum) through special mechanical gates made just for them (TAP, for “transporters associated with antigen processing”). There, another cleaving machine starts dismantling them one link at a time. Crowded nearby are MHCs looking for the unique nine-unit pieces that fit them just right. When no match is found, the chopper keeps cutting the links all the way down for recycling. But if an MHC finds a nine-unit sausage that matches perfectly, it mounts it and ferries it to another special porthole on the cell surface, where it plants it to wave in the breeze. A killer T cell, roving about with a nose that makes a bloodhound look like a man with a bad cold, sniffs all these pieces of meat and is able to detect foreign meat (viral protein scraps) that are not “USDA approved” so to speak. If it finds one, the penalty is severe: the whole cell is targeted for incineration. But it’s a small price to pay for health of the body. This is a nonstop state of war and the stakes are high. Any cell that harbors terrorists must be destroyed. Besides, there are trillions more cells that can take their place.

    What these scientists found was the chopper (aminopeptidase) in the subway that trims the sausages down. They named it ERAAP, and found that it does not need to be concerned with fitting each nine-link sausage to the appropriate meatpole (MHC); it just chops away, one link at a time, and if an appropriate meatpole is nearby to grab it, fine. If not, it chops it all the way down and the individual links (amino acids) are made available for recycling into new proteins. In his News and Views perspective on this discovery, Hans-Georg Rammensee calls this “Survival of the Fitters” – “the way ERAAP works is a fine example of how nature uses the survival-of-the-fittest principle, even inside the cell, to solve a complex task in an economical way.”

    Cell Beats Computer: 100 Trillion Times Faster at Folding a Simple Protein 10/15/2002
    Researchers at Los Alamos National Lab modeled the folding of a “simple” protein of 18,000 atoms on their computers, reports EurekAlert. It took 6 months on 82 parallel processors, which amounts to 34 years of CPU time. The cell folds this particular protein in about 10 microseconds (millionths of a second), which is 100 trillion times as fast. That’s proportional to one second vs. 3.4 million years. The computer algorithm the scientists designed “relies on exhaustive sampling of protein configurations and utilizes massively parallel computing combined with molecular dynamics and a random-sampling Monte Carlo simulation of the thermodynamics.” It is expected that the processing time would grow exponentially with the increasing length of the protein chain, but cells routinely fold their proteins within milliseconds to microseconds. University of Florida reports a record holder: a short 20 amino acid protein that folds within 4 microseconds. Biophysicist Stephen Hagen asks, “What is it that’s special about these molecules that enables them to solve a very difficult computational problem spontaneously in such a short amount of time?”

    Update 10/21/2002: Nature Science Update reports that a scientific team predicted a protein fold successfully by using spare time on 200,000 home PCs in a distributed project called Folding@home. This amounted to about to 2,000 years of computer time. The article states, “Trying to anticipate how the many atoms within a protein interact as it crumples up is a mind-bending problem – involving near a billion steps. Like entering a maze, the molecular backbone can start looping up in a [sic] numerous different ways, yet most paths lead to dead ends.” Somehow the real protein avoids the pitfalls and finds shortcuts through the maze, achieving its correct shape in five milliseconds.

    Announcing: The Protein Big Bang Theory 10/16/2002
    A paper in the Oct 16 online preprints of the Proceedings of the National Academy of Sciences has an intriguing title: “Expanding protein universe and its origin from the biological Big Bang.” Three biochemists from Harvard, University of North Carolina School of Medicine and Boston University attempted to demonstrate a “possible origin of all proteins from a single or a few precursor folds a scenario akin to that of the origin of the universe from the Big Bang.” A striking characteristic of biological proteins is that many have similar folds even with unlike sequences of amino acids; these are called “orphans” because they are nonhomologous: i.e., they appear to have no common ancestors. To explain this, previous investigators imagined that there might be some kind of designability principle that made evolving proteins converge on these special folds. This team set out to show that biological proteins could have diverged instead from simple random precursors; in other words, divergent evolution rather than convergent evolution produced the protein domain. To do this, they graphed all known protein folds on what they call a “protein domain universe graph” (PDUG), tweaked in such a way as to make it scale-free. (An example of a scale-free network is the world-wide web.) After differentiating it from random networks, they deduced that it could have grown by divergent evolution, as proteins evolved through recombination, duplication and mutation, such that folds were preserved even as the sequences were shuffled:

    “It is quite suggestive that the origin of the observed scale-free character of the PDUG lies in the evolutionary dynamics of protein fold genesis as a result of divergent evolution from one or a few precursor domains. To this end, we develop a minimalistic model that aims to explain the scale-free PDUG. Specifically, we assume, as do several other models, that new proteins evolve as a result of an increase in the gene population primarily by means of duplication with subsequent divergence of sequences by mutations, as well as more dramatic changes such as deletions of certain parts sequences and even possible reshuffling of some structural elements (foldons).”

    Their analysis yielded a striking number of orphans, as expected, giving them confidence in their analysis. They caution, however, that the picture is oversimplified:

    “The divergent evolution model presented here is a schematic one, as it does not consider many structural and functional details, and its assumptions about the geometry of protein domain space in which structural diffusion of proteins occurs may be simplistic. However, its success in explaining qualitative and quantitative features of PDUG supports the view that all proteins might have evolved from a few precursors.”

    They conclude by also cautioning that their graphical analysis was just an algorithm selected to “spy” on nature, not that nature used any algorithm to create the protein domain. They chose the algorithm and set the threshold values to attempt to discern natural processes from random ones.

    Ancient Cell Wiser than Most Computer Users 10/23/2002
    The agony of delete strikes many computer users who neglect to back up their data, but an ancient one-celled organism apparently has the wisdom to keep backup copies of its genome. That’s the implication of a story in the BBC News about Tetrahymena, a primitive protozoan that has a macronucleus with the working genome and a micronucleus with a master backup copy. Martin Gorovsky of the University of Rochester has studied this ancient lifeform’s strategy to protect its DNA:

    “Gorovsky’s team believes that in evolutionarily ancient times, cells had to fight against a variety of assaults just as they must today: viruses attacked cells, injecting their DNA to disrupt normal cell functions; and transposons, bits of nomadic genetic material that insert themselves into the cell’s DNA causing havoc. To survive, cells evolved a correction system that recognized the invading DNA and either eliminated or silenced it.”

    The team found that Tetrahymena inspects its DNA against the master copy before passing it on, to ensure the progeny get an uncorrupted copy. Gorovsky suspects a similar defense mechanism is at work in higher organisms.

    Plants Borrowed Membrane Channels 10/25/2002
    All living cells have specialized membrane channels that allow certain molecules in and keep others out; for water, they are called aquaporins (AQPs); for glycerol, they are called aquaglyceroporins (GLPs). There are also ion channels for chloride or potassium. The set of channel families are called membrane intrinsic proteins (MIPs). An international team of researchers has compared the channel proteins from plants, bacteria, and animals, and deduced that plants got their glycerol channels by horizontal gene transfer, with subsequent modification by functional recruitment:

    “The molecular phylogeny of MIPs supports that glycerol transporting in plants was acquired by horizontal gene transfer and functional recruitment of bacterial AQPs. It is likely that these events were triggered by the absence of a GLP homolog in the common ancestor of plants. We find that plant NIPs and GLPs share convergent or parallel amino replacements needed to transport glycerol and therefore represent a remarkable example of adaptive evolution at the molecular level.”

    Their paper is published in the Oct. 23 preprints of the Proceedings of the National Academy of Sciences.

    Fix the Textbooks: Cyanobacteria Weren’t the First 10/25/2002
    “Get ready to rewrite those biology textbooks - again,“ begins the article on EurekAlert based on a story from the Geological Society of America, entitled “Evolution upset: Oxygen-making microbes came last, not first.” A researcher named Carrine Blank from Washington University found that cyanobacteria are too highly evolved to have been the first critters. But “If Blank is correct, her revised evolutionary history of the bacteria raises a difficult question: If cyanobacteria came later, where did the Earth’s earliest oxidants come from which produced banded iron formations?”

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    Elegant, Intricate, Remarkable Describe Cell Channels 09/05/2002
    Gary Yellen of Harvard, in a review article in the Sept 5 Nature waxes prosaic while describing the voltage-regulated channels in cell membranes: with words like “remarkable” and “elegant” he describes the ongoing research into the gatekeepers of the cell:

    “The remarkable optimizations of these channels for permitting rapid and selective ion flow across the hostile barrier of the cell membrane are now mostly apparent, as is the basic repertoire of conformational changes used to gate this flow. We now see the outlines of two general approaches used by intracellular sensor domains to manipulate the channel gates, and some tantalizing details of the transmembrane voltage sensor itself. “

    The potassium channel is capable of passing millions of potassium ions per second through its “selectivity filter” while keeping out sodium ions, smaller and with the same charge. How it does this is on the leading edge of research. Apparently several mechanisms are involved. The channel is able to mimic the arrangement of water molecules that naturally assemble around the ions, and guide them through the channel single file. Also, parts of the channel rapidly flex open and closed in response to the ions or to voltage sensors. A delicate balance of electric charge is required to attract the ions yet prevent them from sticking in the channel. Several ions at a time can be passing through, each validated by the selectivity filter. All this is regulated by voltage sensors, still poorly understood. Cell signalling and nerve impulse transmission rely on the quick and accurate electrical transduction performed by these tiny gatekeepers. An additional wonder is how the channels cooperate with each other, so that an electrical impulse lasting just a few milliseconds can be transmitted at a speed of several meters per second from your toe to your brain. “Electricity plays an unavoidable role in biology,” Yellen opens his article, as he describes how animal cells “have made the management and production of electrical signals into a high art.”

    Peering Into a Tiny Machine on Which the World Depends 09/06/2002
    Sometimes it’s the little things that count. World food supply, ecology, biodiversity are big things, but they depend heavily on a tiny molecular machine called nitrogenase. This machine is worth its weight in gold and is the envy of chemical and structural engineers, but it makes its home in the lowliest of organisms, little bacteria that live around the roots of plants. Its job? To break apart the triple bonds of atmospheric nitrogen molecules and make them available as ammonia to plants, which use this valuable fertilizer to produce proteins for the entire food chain. Molecular nitrogen (N2), though plentiful in the atmosphere (78%) is useless until “fixed” by breaking it apart and combining it with hydrogen as ammonia (NH3). Given plenty of water, nitrogen is the usually the limiting factor in agricultural food production. About half the world’s ammonia is produced by these tiny machines. A little is fixed naturally by lightning, indicating the high energy required. The rest, manufactured by man in the expensive Haber-Bosch process, consumes an estimated 1% of the world’s annual energy output. Those triple bonds are tough nuts to crack! How do the nitrogen-fixing bacteria do it so efficiently, at ambient temperature and pressure? Whoever figures this out and imitates the process will enrich the world’s food supply and save trillions of dollars.

    Scientists have been sharpening the focus more and more on this tiny protein machine, nitrogenase. They knew that precisely placed metal ions (iron, molybdenum) form a critical structure in the heart of the enzyme. They knew other proteins spend ATP to donate electrons to the nitrogen. Now, writing in the Sept. 6 issue of Science, a team of American scientists has sharpened the focus down to 1.16 angstrom resolution. One surprise was that they detected another atom, possibly atomic nitrogen, deep in the heart of the active site. How it gets there, and what role it plays, is still a mystery, but this is another important piece of the puzzle. In the same issue of Science, Barry Smith summarizes the work and concludes, “Once again, nitrogenase has surprised us.”

    More Complex Than Anyone Ever Dreamed: Cell Quality Control 09/09/2002
    According to NewsWise, biochemists like Lynne Maquat at the European Molecular Biology Organization are looking into tinkering with the cell’s quality control system to see if certain error-correcting mechanisms can be switched off. This might provide a means of testing new drugs or treating genetic diseases. In discussing the work, the article uses superlatives to describe how the cell usually corrects mistakes :

    “...mistakes, which are eliminated by dogmatic quality control. ... mRNA molecules are like messengers in a factory, taking a blueprint and then heading to the floor and gathering a team to get the job done. Sometimes, though, the mRNA doesn’t quite get the message right. One common error happens when an mRNA molecule harbors a “stop” or “nonsense” signal before a protein has been completely made. Enter the body’s quality-control system. ... nonsense-mediated decay targets what scientists call a “pioneer round of translation,” during which the body actually produces a kind of rough draft of a protein before giving the go-ahead to the mRNA molecule to begin mass production. ... mRNA puts together an extensive tool kit of molecular machinery to evaluate whether it should pass muster as a legitimate template for proteins. ...
    The identification of such “tool kits,” groups of molecules working together to achieve a task, keeps hundreds of lab groups like Maquat’s around the world constantly busy. Far from the simple and bland “DNA to RNA to protein” sequence of events that many people learn in high school, nearly every cell in the body embodies an incredibly complex construction site where tens of thousands of proteins work in tandem, snipping and cleaving molecules, removing “introns” and splicing together “exons” in various combinations, recruiting molecules to the site, and ferrying molecules over to ribosomes for assembly into proteins. ... “There’s an incredible amount of activity in a small space,” says Maquat, who is secretary/treasurer of the RNA Society and who organized a meeting this summer on the topic of mRNA decay for the Federation of American Societies for Experimental Biology. “A single gene can result in many different proteins depending on how its encoded precursor mRNA is processed; we now know that more than half of human genes can make more than one protein. But with this wonderful flexibility often comes mistakes. The situation is turning out to be more complex than anyone ever dreamed. The degree of RNA processing that the cell undertakes is truly amazing.” The idea of trying to bypass the body’s mRNA surveillance system is formidable. Maquat notes that the system is necessary for survival, and that without it, bad mRNA would create even more instances of disease.”

    Though a formidable prospect, she and her team hope that by allowing some mRNAs to sneak past the quality control guards, some genetic diseases might be treatable, and the process might open up “new vistas for pharmaceutical companies.”

    The Spliceosome: The Most Complex Cellular Machine Yet 09/12/2002
    A molecular machine with 4 RNAs and 145 proteins: that’s the spliceosome, writes a team of Harvard biochemists in September 12 Nature. Its job? “The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins.” Why higher organisms have so many introns (non-coding regions of DNA) and smaller exons (coding regions), and how the exons are joined, is on the cutting edge of DNA research. Formerly considered “junk DNA,” the introns seem to play an essential role in gene expression. They also may provide flexibility for coding regions to join in multiple ways, extending the information content of the DNA. In any event, the splicing of exons together correctly has little tolerance for error, and the spliceosome helps ensure that an accurate messenger RNA gets built before being sent to the ribosome, where the protein product will be assembled. “...we identify 145 distinct spliceosomal proteins,” they announce, “making the spliceosome the most complex cellular machine so far characterized.” Furthermore, the authors find that this machinery is highly conserved (unevolved) between yeast and metazoans [multicellular organisms], including humans:

    “The potentially greater complexity of the human spliceosome is not unexpected in light of the vastly greater complexity of splicing in metazoans compared to yeast. Indeed, most metazoan pre-mRNAs contain multiple introns, the introns are typically thousands of nucleotides, and the splicing signals are weakly conserved. Superimposed on this complexity is the high frequency of alternative splicing, which is in turn further complicated owing to regulation. Thus, many of the metazoan-specific proteins may play roles in the accurate recognition and joining of exons.

    Another paper by German biochemists in the same issue of Nature announces a newly-found role of a chaperone protein named L23. This protein sits at the exit tunnel of the ribosome and forms a docking station for other chaperone proteins, which then grab the emerging polypeptide and fold it properly into its unique shape to become a functioning protein.”

    In the following week’s issue of Nature (Sept 19), Canadian scientists found evidence of introns and splicing machinery in a primitive eukaryote, adding more evidence that spliceosomal introns “are likely to have arisen very early in eukaryotic evolution.”

    Here we see another complex molecular machine, composed of nearly 150 coordinated parts, that operates with skill and precision. “Spliceosomes undergo multiple assembly stages and conformational changes during the splicing reaction,” say the authors, indicating that these machines have many moving parts. They conclude with an acknowledgement that the cell is a veritable factory of complex machinery:

    “The observation that the spliceosome is associated with numerous proteins that function in coupling splicing to other steps in gene expression provides compelling evidence for the emerging concept of an extensively coupled network of gene expression machines.”

    Genetic Code is Even Parity 09/12/2002
    Did you ever learn about even and odd parity in computer class? If so, you know that parity bits are often added to computer codes to reduce errors. If the receiving end reads a byte that is odd when it is supposed to be even, it knows there has been an error. Dónall Mac Dónaill, a chemist at Trinity College Dublin, thinks that DNA uses this technique in the genetic code. He asked why, of all the possible nucleotides, DNA only uses A, C, T, and G. Examining the molecules, he noticed that these four seem to have “even parity.” This makes them very unlikely to pair with the wrong base. An Oxford computational chemist thinks this is a potentially fruitful concept: “It is a novel idea which should provoke others to explore aspects of informatics in the genetic code,” says Graham Richards. The story is summarized on Science Now, and also on Mac Dónaill’s website, and Nature Science Update explained the idea on their site on Sept. 18, emphasizing that “The consequences of wrongly read or copied information can be disastrous. Malfunctioning genes can cause diseases and defects.”

    Primordial Soup Cannot Tolerate Salt 09/17/2002
    In what appears to be a devastating blow to beliefs that life first appeared in the oceans, scientists at UC Santa Cruz, publishing in the journal Astrobiology Vol 2. No. 2 (2002) have experimented with what salt does to RNA and membranes. They found that sea salt destroys fatty-acid membranes and prevents RNA from forming chains (polymerizing), even at concentrations seven times weaker than in today’s oceans. The ingredients of sea salt are very effective at dismembering membranes and preventing RNA units (monomers) from forming polymers any longer than two links (dimers). Noting the “exceptional properties of contemporary cellular membrane structures,” they emphasize that without some kind of osmotic control, primitive vesicles would have collapsed in the presence of divalent cations such as are present in sea salt. Even if early oceans were far less salty, the prebiotic compounds would have needed to be concentrated. But as they logically point out, “Concentrating mechanisms often have a drawback in that they are not selective. That is, not only monomers but also any ionic solute present will be concentrated,” including the damaging salts.

    Considering their study a “crucial piece of information” for origin of life studies, they conclude that the origin of life in the oceans would not be possible, and that a very protected environment of fresh water on the continents would have been necessary for emergent life to evolve far enough to protect itself from the damaging effects of sea salt: “In this very protected environment, simple protocellular entities could thrive until the evolutionary appearance of a primitive metabolic machinery and active salt transport systems in membranes allowed them to overcome the disruptive impact of more saline environments.” The paper is entitled, “Influence of Ionic Inorganic Solutes on Self-Assembly and Polymerization Processes Related to Early Forms of Life: Implications for a Prebiotic Aqueous Medium,” by Monnard, Apel, Kanavarioti and Deamer.

    Motors in Your Ear Amplify Sound 10,000-Fold 09/19/2002
    What limits the hearing range in the ear? Apparently not the eardrum or bones of the middle ear, but the cochlea in the inner ear. We reported in February about prestin, the speedy molecular motor that is involved in controlling the volume of sound on the hair cells of the cochlea. Now, scientists writing in the Sept. 19 issue of Nature have confirmed that prestin is the primary agent in the control of sound amplification, or at least that no other mechanism is necessary to explain the observations. In their research paper entitled, “Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier,” they explain that this little molecular motor, that affects the stiffness of outer hair cells responding to sound waves, provides a 40-60 decibel increase in sensitivity of the ear: a factor of one to ten thousand. By knocking out the prestin motor in mice, the scientists observed a 10,000-fold reduction in hearing sensitivity.

    Scientists Fold a Small Protein 09/25/2002
    According to Nature Science Update, scientists were able to calculate the fold of a small protein of 20 peptides just from knowing its amino acid sequence. The synthetic protein folds into a tight structure like real proteins; “most short chains remain loose and floppy,” the report states. The team used computer simulation to predict the fold that actually occurred, measured by nuclear magnetic resonance spectroscopy. The difficulty in predicting the fold grows exponentially, however, with length of the sequence, and most proteins are hundreds of amino acids long. The precise fold of a protein is essential to its function. Understanding and predicting protein folding from just the amino acid sequence is one of the most formidable challenges facing biochemists. On a related subject, a paper in the Sep. 25 preprints of the Proceedings of the National Academy of Sciences discusses some of the many diseases that occur when proteins do not fold correctly.

    Shipping Labels Used on Cell’s Cargo 09/26/2002
    Bound for New York? Read the label. Destined for the trash can? Read the label. Just as Federal Express or any other shipping company depends on labels to keep myriads of packages on target to equal myriads of destinations, the cell tags its cargo with molecular labels to keep everything on track. Nature (Sept. 26) has two articles on this topic that explain how the cell does it. In The Making of a Vesicle, Anne Schmidt describes work by Ford et al on a protein tag called epsin that stimulates a membrane to curve around, or “package” a piece of cargo for shipment, such as nutrient uptake or removal of parts from the cell surface.

    In another News and Views article, Keith Wilkinson in “Unchaining the condemned” describes how the cell labels obsolete cargo for the recycle bin. Apparently, the protein tag called ubiquitin (which is truly ubiquitous in all eukaryotic organisms) tells the proteasome (the recycle bin) that this cargo is ready for dismantling and salvage. Wilkinson explains:

    “To carry out their functions properly, the proteins in our cells must be in the right place at the right time, and at the right concentration. So it’s vital that cells achieve the correct balance between protein synthesis and destruction. Although we understand much about how proteins are made, it is only in the past ten years that we have come to appreciate the complexity of their degradation. Like everything else, proteins outlive their usefulness and, whether damaged or just no longer needed, they are often condemned to destruction by the covalent attachment of another protein, called ubiquitin. When this process fails, it has profound consequences for events such as cell division, gene expression and the development of cancer.”

    Wilkinson presents the work of Yao and Cohen that indicates that one ubiquitin tag means sort, and several means recycle. The rest of the cell must understand the tag to know what to do, and the proteasome (shaped like a narrow tunnel) has to remove the labels before doing its grisly work.

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