The Ribosomal Function Comes into View

For this article, Eugene Russo interviewed Harry Noller, professor of molecular biology, University of California, Santa Cruz; Venki Ramakrishnan, group leader, Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, UK; and Thomas Steitz, a professor of molecular biophysics, biochemistry, and chemistry, Yale University. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. N. Ban, P. Nissen, J. Hansen, M. Capel, P.B. Moore, T.A. Steitz, "Placement of protein and RNA structures into a 5 angstrom-resolution map of the 50S ribosomal subunit," Nature, 400:841-7 Aug. 26 1999. (Cited in 160 papers) J.H. Cate, M.M. Yusupov, G.Z. Yusupova, T.N. Earnest, H.F. Noller, "X-ray crystal structures of 70S ribosome functional complexes," 285:2095-104, Science, Sept. 24, 1999. (Cited in 209 papers) W.M. Clemons, J.L.C. May, B.T. Wimberly, J.P. McCutcheon, M.S. Capel, V. Ramakrishnan, "Structure of a bacterial 30S ribosomal subunit at 5.5 angstrom resolution," Nature, 400:833-40, Aug. 26, 1999. (Cited in 160 papers) ©2001 AAAS Crystal structure of the Thermus thermophilus 70S ribosome, viewed at the subunit interface (Reprinted with permission from Science 292:883-896, © 2001 AAAS) Translation is among the most fundamental of all biological process. Yet even after four decades of research stretching into the 1990s, scientists couldn't agree on any single model that depicted exactly how this RNA-orchestrated process of protein synthesis takes place. Investigators inevitably turned to the only method that could precisely capture the fine detail of the actual structures involved: X-ray crystallography. "A lot of biochemical and genetic data had been generated," says Venki Ramakrishnan, group leader, structural studies division, Medical Research Council Laboratory of Molecular Biology, UK. "But it was hard to make sense of that data, and hard to go forward without a structure." A better understanding of ribosomes, the assembly plants for translation, was essential for attaining a clearer picture of the set of biochemical events involved in the process. More specifically, researchers needed to elucidate the interaction of the two subunits, 30S and 50S, that make up the ribosome. These Hot Papers helped piece together this complex puzzle: The Clemons et al. paper reported the structure of the 30S subunit in the bacterium Thermus thermophilus; the Ban et al. paper detailed the 50S subunit in the bacterium Haloarcula marismortui; and the Cate et al. paper featured a lower resolution structure of the entire ribosome, also in T. thermophilus. Ribosomal structures are presumed similar across species, based on the observed conservation of primary and secondary ribosomal RNA structures. Although they did not provide the complete structures, these papers, "signaled that the problem had been cracked and that it would eventually be solved," contends Ramakrishnan. Subsequent papers published in 2000 provided the two subunits' correct and complete structures;1-3 a 2001 paper reported a higher resolution structure of the entire ribosome.4 This improved resolution greatly advanced scientists' understanding of the complex's biochemical characteristics. Piecing the Puzzle Together Studies prior to the 1999 papers had hinted at the location of parts of the ribosomal complex, how those parts fit together, and the sorts of changes that occur when the ribosome alters states. "But it was hard to make sense of it all because there was no feeling for how this was all organized in three dimensions," explains Ramakrishnan, senior author of the Clemons paper. Electron beam-powered electron microscopy (EM) work on the ribosome had its limits. "The EM stuff was just a big blob at that time," says Thomas Steitz, a professor of molecular biophysics and biochemistry and chemistry at Yale University. Steitz, the Ban paper's senior author, notes that until a few years ago, researchers did not see the application of X-ray crystallography to the ribosome as feasible. Proof of principle results from Steitz's group in 1998 showed otherwise.1 "The techniques haven't changed over the last three years," says Steitz. "It's the sense that here's something that can be done if you do it right." The ribosomal subunits' movements, relative to one another, have long been thought to play a major role in translation. Researchers know that the subunits somehow come apart at the end of the message and recombine at its beginning, part of an elegant and crucial partnership. The subunits work together to move the transfer RNA and associated messenger RNA by precisely one codon. However, each subunit also has a more specialized role. As amino acids are added to the protein chain, peptide bonds form at the 50S subunit, which is twice as big as the 30S. The 30S is responsible for making sure that only the correct tRNAs, those possessing the right anticodons, bind to complementary mRNA codons to ensure an accurately translated genetic message. "We understand that quite a lot about the match between codon and anticodon is recognized as a result of these structures," comments Ramakrishnan. He notes that the exact mechanism by which the 30S recognizes the tRNA was first hinted at in an updated, high-resolution structure reported in 2000.2 Follow-up work from Ramakrishnan's group has shown the intricate binding behavior of six different antibiotics, including the clinically common streptomycin and tetracycline.3 Researchers could not initially make as detailed an interpretation about the RNA interactions of the larger, more complex 50S structure, explains Steitz. "One had no idea of what it might look like in molecular terms," he says. For example, Steitz's team wondered whether the proteins affiliated with the 50S subunit surrounded it like a virus, or were located within the subunit (like a nucleosome). As it turns out both are true. Steitz emphasizes that the 1999 paper says nothing about the biochemical mechanism of protein synthesis. Those details came later in a pair of papers by Steitz's group,4,5 which themselves have been cited 170 and 110 times, respectively (Web of Science, ISI, Philadelphia, Pa.). The more recent papers report that substrates bind to the subunit, allowing researchers to sort out the structural constraints of the mechanism of peptide-bond formation. "At the lower resolution, we could see nothing like that," comments Steitz. "We could just say, 'Well, there's a lot of RNA in there, it's tightly packed, proteins are interspersed.'" Detailing the Ribosomal Structure Courtesy of Harry Noller Harry Noller A group led by Harry Noller, a professor of molecular biology, University of California, Santa Cruz, reported the entire ribosomal structure. Noller's group saw the whole organelle for the first time, including the tRNAs' binding sites. As part of more detailed work published this year, Noller's team, with the help of the atomic coordinates of each of the subunits reported in the 2000 papers, showed exactly how the tRNAs interact with the inter-subunit bridges.6 In fact, some of these bridges were apparently part of the tRNA binding sites. Noller thinks this observation may help address what he calls one of the complex's most interesting mysteries: how the huge tRNA molecules move inside the ribosome. "The inescapable implication is that the ribosome is actually a molecular machine with moving parts," explains Noller. "And it's moving these things through some kind of molecular mechanical mechanism." He believes that the bridges between the subunits are in some way connected to the molecular movements that propel the tRNA and the attached mRNA through the ribosome. "That's really one of the most exciting things for us," says Noller. "It's like we've been wondering how the car works, and now you pop the hood for the first time and you're looking at the engine." Steitz adds that at least six labs are working on examining the different ribosomal states. Unfortunately, the laborious task of X-ray crystallography captures only one frame of this dynamic process at a time. A much faster but lower resolution technique called cryo-electron microscopy (cryo-EM) may be key to constructing a detailed "movie" of ribosomal activities. Cued by cryo-EM, which relies on frozen solutions of ribosomes rather than crystallized proteins, researchers might acquire and then piece together many low-resolution, X-ray crystallography snapshots of the complex.7 Eugene Russo (erusso@the-scientist.com) is a contributing editor for The Scientist. References 1. N. Ban et al., "A 9 angstrom resolution X-ray crystallographic map of the large ribosomal subunit," Cell, 93:1105-15, 1998. 2. B.T. Wimberly et al., "Structure of the 30S ribosomal subunit," Nature, 407:327-39, 2000. 3. D.E. Brodersen et al., "The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit," Cell, 103[7]:1143-54, Dec. 22, 2000. 4. N. Ban et al., "The complete atomic structure of the large ribosomal subunit at 2.4 angstrom resolution," Science, 289:905-20, 2000. 5. P. Nissen et al., "The structural basis of ribosome activity in peptide bond synthesis," Science, 289:920-30, 2000. 6. M.M. Yusupov et al., "Crystal structure of the ribosome at 5.5 angstrom resolution," Science, 292:883-96, May 4, 2001. 7. J. Frank, et al., "A ratchet-like inter-subunit reorganization of the ribosome during translocation," Nature, 406:318-22, 2000.

The Ribosomal Function Comes into View

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