The Chemical Basis for Life the 2009 Nobel Prize in Chemistry
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The chemical basis for life The 2009 Nobel Prize in Chemistry. Part 2 PETER KARUSO The story of the 2009 Nobel Prize in Chemistry is an epic in time and proportion. The second instalment focuses on the final leg of the race for the ribosome and the contri- butions of Tom Steitz, Venki Ramakrishnan and others. he year 1999 was pivotal in the race for the struc- Venkatraman Ramakrishnan Tture of the ribosome – the cell’s largest ‘machine’. Venkatraman Ramakrishnan was born in At the Ribosome Conference in Helsingor, Denmark, Chidambaram, Tamil Nadu, India, in 1952. He in June 1999, several groups revealed their results for obtained his PhD in physics in 1976 from Ohio the !rst time. Peter Moore, Tom Steitz, Venki University, USA. As a student he was much more Ramakrishnan, Harry Noller and Ada Yonath gave interested in his monthly deliveries of Scienti!c Amer- consecutive talks at the meeting. Yonath described ican than his PhD project. He thought the really her results on the 30S subunit but her low-resolution interesting stuff was in the biomolecular sciences results paled in comparison to those of Moore and rather than physics so planned to switch. Ramakr- Steitz, and Noller. However, everyone was amazed at ishnan ended up in Peter Moore’s lab (Chemistry, the results presented by someone who had not previ- Yale) and worked on small-angle neutron scattering of ously been a player in the ribosome area: a dark horse the ribosome from 1978 to 1982. He continued his (Ramakrishnan) had scooped everyone with a 5.5 Å interest in the ribosome at the Brookhaven National resolution structure of the small (30S) ribosomal Laboratory and then at the University of Utah where subunit. The structure was tantalisingly close to he solved the structure of about six individual ribo- atomic resolution (3–3.5 Å). The race for the high- somal proteins and became interested in the structure resolution structure of the ribosome was now well of the entire 30S subunit, which plays a crucial role in and truly on. accepting or rejecting the amino acid-carrying tRNA. Image credit: Neil Grant MRC-LMB Cambridge. From Yonath’s presentation at the international ribosome meeting in 1995 (Canada), he knew that no one had yet been successful in producing good crystals of the 30S subunit, so he decided to try. His team made steady progress, even though he moved across the Atlantic from Utah to Cambridge in 1999. During that time, though, Ramakrishnan never really let on that he was trying to solve the structure of the ribosome. At the 1999 Denmark meeting, Ramakr- ishnan explained that turning his postdoc (Brian Wimberly; now at Rib-X in New Haven) loose on the structure of the ribosome was like giving the keys of a Ferrari to a 24 FEBRUARY 2010 teenager. Not only was he able to identify key land- !t’ hypothesis of enzyme mechanisms over the ‘lock- marks in the diffraction patterns but was able to and-key’ mechanism that had dominated since Paul discern the path of the RNA through the 30S Ehrlich (Nobel Prize in Physiology or Medicine, subunit. In August 1999, Ramakrishnan’s team 1908). He became interested in the structure of the published the !rst structure of the small subunit in ribosome more recently, through his friend and Nature (Clemons et al. 1999) with 5.5 Å resolution, colleague Peter Moore (Sterling Professor of Chem- while the team of Peter Moore and Thomas Steitz at istry at Yale University). Interestingly, like Steitz, Yale University described the structure of the larger Moore obtained his PhD from Harvard in 1966 and subunit (Ban et al. 1999) in the same issue to 5 Å had a postdoctoral position at the MRC Laboratory resolution. In a series of papers from 2002 to 2005, of Molecular Biology in Cambridge at the same time Ramakrishnan began to explore the !ne details of as Steitz but obtained a position at Yale a year earlier. how the ribosome works. What emerged was a simple Moore has focused his entire career on under- and coherent explanation for a number of features standing the mechanism, structure and function of related to codon usage and proofreading of the the ribosome. He has made elegant use of a wide emerging protein that had been poorly understood up variety of biophysical techniques including small until then. angle neutron and X-ray scattering, solution-state NMR spectroscopy and other biophysical techniques Tom Steitz to determine the atomic structure of several ribo- Thomas Steitz was born in Milwaukie, Wisconsin somal RNA sequences. However, his most signi!cant (1940) and attended Wauwatosa High School where contributions came in collaboration with Steitz, a his interest in chemistry was already obvious. He went consummate crystallographer. on to study chemistry at Lawrence University and Moore and Steitz initially had major technical then a PhD at Harvard in Biochemistry and Molec- problems in producing good crystals of the ribosome. ular Biology (1966). Coincidentally, he spent a post- It took them 2 years to work these out, but once they doctoral period at the MRC Laboratory of Molecular were solved, progress was rapid. By June 1998, they Biology, Cambridge (1967–70), where Ramakr- has a 9 Å resolution structure of the large (50S) ishnan now works, and then obtained a position at subunit, reaching 5 Å resolution by 1999 (Ban et al. Yale, where he has been ever since. His research was 1999) and then an amazing 2.4 Å by 2000 (Ban et al. primarily focused on the mechanisms of enzymes and 2000). This rapid progress was made possible by an his work was instrumental in supporting the ‘induced innovation by Steitz. He had solved the phase problem in crystallography of large Image credit: Michael Marsland, Yale University. complexes, not by labelling proteins with clusters of heavy atoms as Yonath had tried, but by combining X-ray crystallography with electron microscopy (EM). Cryo-EM is a technique that uses the rather low- resolution images generated by elec- tron microscopy of proteins frozen in ice and combines thousands of these images to produce a relatively high- resolution image. The technique had been around for two decades but it was not until the mid-1980s when Jacques Dubochet (European Molec- ular Biology Laboratory in Heidel- FEBRUARY 2010 25 berg, Germany) had developed a way of rapidly freezing molecules in water so that they ended up encased in vitreous ice (McDowall et al. 1983) that the technique was able to generate high-resolution images. At about this time, Miloslav Boublik (Rocke- feller University) showed Joachim Frank (Columbia University) some electron microscopy images of the human ribosome. This is when he came up with the Figure 1 The mechanism of peptide bond formation in the idea of combining many low-resolution micrographs ribosome involves a proton shuttle, NH to OH and OH to O on to create one high-resolution image. Frank produced the RNA, proving that the ribosome is, in fact, a ribozyme. the !rst images of the human 40S subunit using this technique at 20 Å resolution in 1981 (Frank et al. also revealed the efforts of his group (Cate and the 1981). These results sparked a lot of interest and Yusupovs). They had managed to get 7.8 Å resolution started another race – primarily between Frank and of the complete ribosome (Cate et al. 1999). This Marin van Heel (Fritz Haber Institute of the Max relatively low-resolution map of the whole ribosome Planck Society, Berlin) for the structure of the ribo- was very signi!cant because it gave the !rst hint to some by cryo-EM but also between the crystallogra- how the two ribosomal subunits interact and phers and cryo-EM community in general for atomic con!rmed Noller’s theory that there is no protein resolution of the ribosome. In 1995, at a Gordon within 18 Å of the ribosome’s active site. Noller went Conference in New Hampshire, van Heel and Frank on to eventually produce pictures of the ribosome at had side-by-side posters with structures of the whole 3.7 Å resolution. In 2007, Noller and Steitz were bacterial ribosome resolved to about 25 Å resolution. jointly awarded the prestigious Gairdner Foundation While cryo-EM eventually was not able to produce International Award for their groundbreaking studies images of atomic resolution, it is true to say that Steitz on the structure and function of the ribosome. wouldn’t have got anywhere without the cryo-EM The high-resolution structure of the ribosome was images. used by Steitz, Moore and Bill Jorgensen (also from Steitz’s images at 2.4 Å resolution of the large Yale Chemistry) to launch a start-up company (Rib- subunit proved unequivocally that the peptide bond X) in 2001. The company has licensed the structural formation is catalysed exclusively by RNA and led information obtained by Moore and Steitz from Yale Moore to postulate the !rst detailed mechanism of to design and synthesise unique inhibitors of the the process (Fig. 1) (Nissen et al. 2000). This proved, bacterial ribosome, starting from information on how beyond a doubt, that the ribosome was a ribozyme, natural products such as paromomycin bind to and something postulated by Harry Noller a decade inhibit speci!cally bacterial protein synthesis. The earlier and which has subsequently been used as company has raised more than US$173 million in evidence of an ‘RNA world’, where the current roles four rounds of venture capitalisation. They currently of DNA and proteins were originally carried out have two drugs (dela"oxacin and radezolid) about to exclusively by RNA.