The Ribosome's 30S Subunit Comes into Focus

For this article, Laura DeFrancesco interviewed Venki Ramakrishnan, group leader, Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, UK; and William Clemons, postdoctoral fellow at Harvard Medical School. 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. B.T. Wimberly, D.E. Brodersen, W.M. Clemons, R.J. Morgan-Warren, A.P. Carter, C. Vonrhein, T. Hartsch, V. Ramakrishnan, "Structure of the 30S ribosomal subunit," Nature, 407:327-39, Sept. 21, 2000. (Cited in 168 papers) A.P. Carter, W.M. Clemons, D.E. Brodersen, R.J. Morgan-Warren, B.T. Wimberly, V. Ramakrishnan, "Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics," Nature, 407:340-8, Sept. 21, 2000. (Cited in 77 papers) Courtesy of Venki RamakrishnanA Pretty Package: The atomic structure of the 30S ribosomal subunit. The molecule consists of 1521 nucleotides of RNA and 20 protein chains. This ribbon diagram shows the major domains of RNA colored in red, green, gold, and cyan, going from the 5' end to the 3' end. The proteins are in contrasting colors. In 1999, X-ray crystallographers did what many considered impossible—they crystallized very large, complicated structures. In that year, Venki Ramakrishnan and colleagues from the Medical Research Council (MRC) Laboratory of Molecular Biology, UK, published the 30S ribosomal subunit's structure at 5.5 Angstrom resolution.1 Around the same time, two other groups published structures of the 50S subunit and 70S ribosome at similar resolution.2,3 Although the resolution was still not sharp enough to solve the structure, the work signaled that a complete solution was not far off. One year later, in these Hot Papers, Ramakrishnan's group described the complete structure of the 30S subunit at 3 Angstrom resolution, both isolated and complexed with antibiotics. This information answered some long-standing questions on protein synthesis and provided insights into the ways antibiotics can foil the process—knowledge that might someday lead to better drugs. During the 1990s, Ramakrishnan says a number of discoveries were made that could have helped other researchers arrive at the same conclusions. For one, it had been almost 10 years since Ada Yonath of the Weizmann Institute showed that crystals of the 50S subunit could be diffracted to high resolution, a feat that broke an important psychological barrier, he says. The second discovery involved synchrotrons, which provide the high energy necessary to diffract such large structures; these were available since 1990. But scientists were using established techniques to clarify the diffraction, with no results. "So it was clear," says Ramakrishnan, "that some new thinking was required to approach the crystallography." Purity Pays Off One key was getting homogeneous crystals. Other researchers had made crystals of Thermus thermophilus 30S subunits that diffracted at 8-10 Angstroms—poor crystals, as Ramakrishnan called them. But when his group realized that some of these crystals completely lacked the S1 ribosomal protein, they devised methods to remove the small fraction of those 30S particles that retained it. "Using these homogenous particles was a prerequisite to obtaining a structure," Ramakrishnan recalls. Curiously, only 30S subunits from T. thermophilus diffracted at better than 5 Angstrom resolution. Harvard Medical School's William Clemons, then a graduate student at MRC, led the group to try other species knowing that 'poor' T. thermophilus crystals weren't up to the task. But these attempts failed and the investigators returned to the T. thermophilus species knowing they could at least get a somewhat clear crystal. Once they had the structure in hand, the researchers could see why the T. thermophilus 30S subunits worked where others did not. The structural analysis of the 30S subunit revealed a feature along its bottom that was shaped like a spur. Sequence comparisons of the 16S RNA from this region showed that this area varies among different species, which is usually interpreted to mean that it does not have much biological importance. However, in the case of the T. thermophilus 30S subunit, the spur had an important function, as far as the crystallographers were concerned. The spur docks into the P site, one of the tRNA binding pockets, essentially locking the 30S down into a specific conformation. According to Clemons, this was the critical difference. "The 30S subunit is very flexible, the top is loosely attached to the bottom," he says. "By having something locked in there, it stabilized [the subunit]." Phase Problems Overcoming the difficulty of creating pure crystals was not the only challenge faced by the MRC group. Another was to solve the phase problem. When X-rays hit a crystal, they are diffracted into many spots called "reflections," each of which represents a wave. Along with the need to know wave intensity (amplitude), researchers also need to know how late or early each wave arrives relative to all the others (the phase of the wave) to solve the structure. Since the 1950s, crystallographers soaked their crystals in heavy atoms to provide some landmarks in the diffraction pattern. But with a structure as large as ribosomal subunits, it wasn't clear whether heavy atoms would give off enough of a signal to provide the phase. Ramakrishnan realized that a special property of certain heavy atoms called anomalous scattering would probably have enough signal. But there was still the question of how to locate these atoms in the structure. The solution to finding these atoms came from a group at Yale University working on the structure of the 50S subunit. In the lab of Thomas Steitz, Sterling professor of molecular biophysics and biochemistry, researchers showed that a super-heavy cluster of 18 tungsten atoms could be located directly from the data and give low-resolution images.4 In turn, they could use these images to locate the other heavy atoms, and thus bootstrap their way to high resolution. Ramakrishnan's group, however, found that their crystals could not withstand the radiation levels required to get high-resolution data. Data from different crystals can be pooled, but only if the crystals are identical which still was not the case with the 30S subunit preparations. Here a more prosaic solution prevailed. The investigators froze more than 1,000 crystals, took quick "test shots" on hundreds of them, and then grouped together all the crystals that were similar and well diffracted. The "Wobble Hypothesis" at Atomic Resolution Before the atomic structure was solved, researchers made educated guesses, using indirect data, to determine the mechanism of the complex series of reactions collectively known as protein synthesis; the exact locations of the various chemical groups that were involved were not known. Courtesy of Venki RamakrishnanVenki Ramakrishnan "Now," Ramakrishnan says, "it is much easier to study mechanisms and interpret biochemical results." For example, as published later,5 the detailed structure has revealed how the ribosome recognizes the correct tRNA, the so-called "decoding" process. Through an ill-defined course of action, the 30S subunit can distinguish among a host of possible tRNAs to select the one that matches the codon on mRNA. How the ribosome recognizes each one had not been known. Ramakrishnan's group discovered that two adenines and a guanine on the 16S ribosomal RNA flip out and read the geometry of the minor groove of the three-base helix formed between the codon and anticodon. These flipped out nucleotides can tell whether the base pairs are Watson-Crick (perfectly matched) or not. Interestingly, the reading is done stringently at the first two positions of the codon, but not at the third (or "wobble") position. With this information, a 35-year-old theory can now be explained at the atomic level. In 1966, Francis Crick theorized the wobble hypothesis to explain how the same tRNA can work with codons that differ at the third position.6 Ramakrishnan says, "We can now see how this can be rationalized by the structure. The ribosome requires Watson-Crick base pairs at the first two positions but is more tolerant at the third." Paromomycin Produces Errors In the second Hot Paper1 Ramakrishnan's group investigated the effects of antibiotic binding on the 30S subunit structure and discerned how these drugs wreaked havoc with protein synthesis. When paromomycin is bound to the 30S subunit, for example, two of the three nucleotides involved with decoding are moved to where they can interact directly with mRNA and tRNA. Repositioning these nucleotides reduces the amount of energy required to make this transition, energy that is usually provided by the hydrogen-bonding made between a well-matched codon and anticodon. In essence, paromomycin pays part of the freight, reducing the amount of energy that must come from the pairing, which permits near-cognate matches to occur, thus increasing the error rate of protein synthesis. Now that the structure of antibiotic complexes with the ribosome can be solved, and the detailed interactions observed, new opportunities for creative drug design are possible: For example, how antibiotics interact with their binding site could be improved, or new compounds could be designed to that site or even to completely new sites that affect function. Says Ramakrishnan,"In that sense, the ribosome has now become like other proteins that are drug targets, because structures are known. Not surprisingly, many companies, both large and small, are interested in this." Laura DeFrancesco (defrancesco1@earthlink.net) is a freelance writer in Pasadena, Calif. References 1. W.M. Clemons et al., "Structure of a bacterial 30S ribosomal subunit at 5.5 angstrom resolution," Nature, 400:833-40, 1999. 2. N. Ban et al, "Placement of protein and RNA structures into a 5 angstrom-resolution map of the 50S ribosomal subunit," Nature, 400:841-7, 1999. 3. J.H. Cate et al., "X-ray crystal structures of 70S ribosome functional complexes," Science, 285:2095-104, 1999. 4. N. Ban et al., "Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit," Nature, 400:841-7, 1999. 5. JM Ogle et al., "Recognition of cognate transfer RNA by the 30S ribosomal subunit," Science, 292:897-902, May 4, 2001. 6. F.H. Crick, "Codon-anticodon pairing: The wobble hypothesis," Journal of Molecular Biology, 19:548-55, 1966.

The Ribosome's 30S Subunit Comes into Focus

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