Atomic Resolution of Large Ribosomal Subunit Reveals Structure

Graphic: Courtesy of Peter Haebel RIBOSOME REVEALEDA view of the active site of the large ribosomal subunit. Proteins of this large assembly are shown in yellow; the ribosomal RNA is in gray. Scientifically speaking, the ribosome's make-up and raison d'etre is elementary: Composed of RNA and proteins, it's the cell site where amino acids get strung together to form new proteins. And, while protein synthesis is a well-studied cellular process, says Nenad Ban, assistant professor, Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zurich, "most ribosomal studies in the past were done 'blindly' since no high-resolution structural information on the ribosome was available." Data derived from the Science Watch/Hot Papers database and 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, P.B. Moore, T.A. Steitz, "The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution," Science, 289:905-20, Aug. 11, 2000. (Cited in 352 papers) P. Nissen, J. Hansen, N. Ban, P.B. Moore, T.A. Steitz, "The structural basis of ribosome activity in peptide bond synthesis," Science, 289:920-30, Aug. 11, 2000. (Cited in 226 papers) That was the case until two years ago. This pair of Hot Papers gave scientists a first-time, atomic-resolution view of the large ribosomal subunit and a mechanism to relate its atomic structure to its function.1,2 "Many of the biochemical, genetic, and biophysical experiments that were conducted were important and revealing but sometimes also difficult to interpret and seemingly contradictory," states Ban. No high-resolution structure of the large ribosomal subunit existed prior to these papers. Ban, along with Poul Nissen, associate research professor, molecular biology department, Aarhus University, Denmark, and Jeffery Hansen, a postdoc in the lab of Thomas Steitz, a Howard Hughes Medical Institute (HHMI) investigator and molecular biophysics and biochemistry professor at Yale University, used cutting-edge X-ray crystallographic techniques they developed to determine the structure. Chemistry professor Peter B. Moore, also at Yale, coauthored the two papers. Ban and Nissen were postdocs in the Steitz lab. Nissen says the papers are better thought of as "one giant paper divided into two." The first paper1 describes the overall structure and its determination. It demonstrates "how the RNA is folded, how the proteins fit into it, what's holding the large subunit together, and the principles of making this very large macromolecular machine," states Steitz. "That was a huge advance for the field," notes Jennifer Doudna, molecular and cell biology professor, University of California, Berkley, and fellow HHMI investigator, because it unveiled what the catalytic center of this major cellular machine looks like. Doudna also investigates RNA structure. Photo: Courtesy of Poul Nissen Poul Nissen The second paper2 focuses on the active site of the large ribosomal subunit, namely the peptidyl transferase activity responsible for peptide bond formation that occurs during messenger RNA-mediated protein synthesis. Says Nissen: "The most important finding from an academic point of view was to see that the ribosome was in fact a ribozyme. In other words: RNA makes protein and this is how gene expression started in evolution." He explains that in the second paper the team proposed that an adenosine residue appeared to interact with the substrate parts involved in peptide bond formation and therefore could be helping this reaction by contributing acid-base catalysis and transition state stabilization during protein formation. Data from the lab of Marina Rodnina in Witten, Germany, and others have confirmed this idea, showing that the adenosine pointed out by the Steitz lab is critical for general base catalysis of peptide bond formation.3 Further studies from the Steitz lab, on the other hand, have contradicted a direct role of this adenosine in transition state stabilization.4 THE RIBOSOME IS A RIBOZYME Steitz and colleagues have been studying ribosomal structure since 1995, steadily figuring out the methods for obtaining X-ray crystallographs of ribosomal subunits. A few years before that, Ada Yonath, member of the Max Planck Institute, Berlin, and Israel's Weizmann Institute, began working with crystals of Haloarcula marismortui. Eventually the Steitz group figured out methods for gaining a high-resolution picture of those crystals. H. marismortui, a member of the kingdom Archaea, is a halophile that lives in the Dead Sea at very high salt concentrations. The rationale for using this and other extremophiles for ribosomal studies is that their ribosomal assemblies are more robust and stable at extreme conditions, and therefore will hold up in experimental manipulations. The notion that the ribosome is a ribozyme, a biological, RNA-comprised catalyst, is "an idea long championed by Harry Noller, Carl Woese and others,"5 wrote HHMI president and 1989 Nobel laureate Thomas Cech. "The way in which we have proven that it's a ribozyme is by showing that it's only RNA in the vicinity of the region where peptide bond formation occurs," explains Steitz. "That's a significant discovery in my view because it absolutely proves that RNA catalysis preceded protein-based enzymes, evolutionarily." What's interesting about RNA, continues Steitz, is that it can have both a genome function, like DNA in storing information in its sequence, and it can be an enzyme, as Cech determined.6,7 "So that's one way of solving the chicken-and-the-egg problem of how do you get an enzyme and a genome if you need an enzyme in order to copy a genome. The answer is: The genome can also function as an enzyme," says Steitz. NEW VIEW Ribosomes are complex aggregations of RNA and protein, and during protein synthesis each one is composed of a small and large subunit.8,9 Steitz's and Moore's high-resolution picture of the large subunit allowed a first-ever view of how the domains of 23S RNA of the large subunit fit together to form a large monolithic structure not unlike the pieces of a three-dimensional jigsaw puzzle. Says Steitz: "I didn't think we knew it would turn out that way and indeed the small subunit has a different structural principle in a sense that the domains are more separate, as is more typical of a protein architecture." The large subunit is more regularly shaped. "I think the other finding that surprised us the most ... was the observation that about a dozen of the subunit's proteins have extended, irregularly folded tails or loops that penetrate into the interior of the RNA," remarks Steitz. "That's not generally observed with proteins. These loops that reach into the subunit's interior are thought to stabilize the structure. These proteins are abundant everywhere on the subunit's surface except at the active site where the peptide bonds are formed, the ribozyme, and the surface that contacts the small subunit." Ribosomes perform several critical functions in protein synthesis. The Nissen paper showed how substrate analogs bind to the ribosome, and how the ribosomal RNA structure is able to bind and orient the receptor ends of transfer RNAs, which bear the amino acids to be strung into a protein. Photo: Courtesy of Eilika Weber-Ban Nenad Ban IMPLICATIONS FOR FUTURE RESEARCH The colleagues suggest many practical and theoretical consequences of their discovery, from the treatment of bacterial infections (ribosomes are targeted by antibiotics) to revamping molecular evolutionary theory. "The structure will allow rational modification of these drugs in order to overcome bacterial resistance, a major problem in today's hospital environments, and to design novel antibiotics," suggests Ban. Steitz, Moore, and colleagues have recently formed the company Rib-X Pharmaceuticals in New Haven, Conn., which will use the ribosome to design antibiotics. The papers also enhanced the research community's knowledge of RNA structure. From the Ban paper, the Steitz group deposited the coordinates of 3,000 nucleotides and 27 RNA-binding proteins into the Protein Data Bank, which increased the overall database of RNA structure by about six-fold. The essential finding that researchers keep coming back to is the confirmation that the ribosome is indeed an RNA enzyme. "We knew that the ribosome had to evolve from RNA, and there was a lot of evidence pointing to that, but seeing that for the first time was very exciting," says Doudna. "And the idea that we're looking almost back in evolutionary time at this incredibly beautiful and complex molecule has stimulated enormous research in the field." Cech put the findings into perspective: "The bacterial ribosome is composed of three RNA molecules and more than 50 proteins. Its key components are so highly conserved among all of Earth's species that a similar entity must have fueled protein synthesis in the common ancestor of all extant life."5 Karen Young Kreeger (kykreeger@aol.com) is a contributing editor. References 1. N. Ban, P. Nissen, J. Hansen, P.B. Moore, T.A. Steitz, "The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution," Science, 289:905-20, Aug. 11, 2000. (Cited in 352 papers) 2. P. Nissen, J. Hansen, N. Ban, P.B. Moore, T.A. Steitz, "The structural basis of ribosome activity in peptide bond synthesis," Science, 289:920-30, Aug. 11, 2000. (Cited in 226 papers) 3. J. Hansen et al., "Structural insights into peptide bond formation," Proceedings of the National Academy of Sciences, 99:11670-5, 2000. 4. V.I. Katunin et al., "Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome," Molecular Cell, 10:339-46, August 2002. 5. T.R. Cech, "Enhanced: The ribosome is a ribozyme," Science, 289:878-9, 2000. 6. K. Kruger et al., "Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena," Cell, 31:147-57, 1982. 7. A.J. Zaug et al., "The intervening sequence RNA of tetrahymena is an enzyme," Science, 231:470-5, 1986. 8. B.T. Wimberly, "Structure of the 30S ribosomal subunit," Nature, 407:327-39, 2000. 9. L. DeFrancesco, "The ribosome's 30S subunit comes into focus," The Scientist, 16[9]:24, April 29, 2002.

Atomic Resolution of Large Ribosomal Subunit Reveals Structure

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Graphic: Courtesy of Peter Haebel
RIBOSOME REVEALEDA view of the active site of the large ribosomal subunit. Proteins of this large assembly are shown in yellow; the ribosomal RNA is in gray.