The ribosome returned

Review The ribosome returned Peter B Moore Address: Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA. Email: Published: 26 January 2009 Journal of Biology 2009, 8:8 (doi:10.1186/jbiol103) The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/8/1/8 © 2009 BioMed Central Ltd Abstract Since the mid-1990s, insights obtained from electron microscopy and X-ray crystallography have transformed our understanding of how the most important ribozyme in the cell, the ribosome, catalyzes protein synthesis. This review provides a brief account of how this structural revolution came to pass, and the impact it has had on our understanding of how the ribosome decodes messenger RNAs. About 20 years ago, for reasons now lost in the mists of the 20th century, I wrote a review about the ribosome for Nature [1]. Ribosomes had been discovered in the mid1950s and, until the late 1960s, ribosome research was a major part of molecular biology. By the late 1960s it had emerged that ribosomes are the polymerases that catalyze protein synthesis under mRNA control. Satisfied with that level of understanding, most who had worked on protein synthesis during the ‘golden age’ of molecular biology sought greener pastures in the years thereafter, and interest in the ribosome waned. The thesis of my review, which was entitled ‘The ribosome returns’, was that the ribosome field was poised for advances so dramatic that it would regain the prominence it had last enjoyed in the mid-1960s. In 1988 there were two reasons for optimism. First, the discovery of ribozymes in the late 1970s had stimulated the interest of biochemists and molecular biologists in RNAcontaining objects generally, and the ribosome is the most important RNA-containing object of them all. Second, the shortage of structural information that had for so long plagued the ribosome field seemed ready to end. A month or so ago, I agreed to write a successor to ‘The ribosome returns’ for Journal of Biology, but shortly thereafter I started having second thoughts. As Yogi Berra is alleged to have said, “It is hard to make predictions, especially about the future”. By writing a successor to ‘The ribosome returns’ I would be in the embarrassing position of calling attention to an ancient review, the very title of which was a prediction. Below I provide a personal account of what happened in the ribosome field between 1988 and 2000 and my assessment of where the field stands today. As it happens, the ribosome did return, but it took a while. How the structural drought ended By 1988, a lot had been learned about the three-dimensional organization of the ribosome. The shapes of the two ribosomal subunits, and of the complex they form during protein synthesis, were known at low resolution (Figure 1), and it was understood that protein synthesis occurs in the gap between the two subunits. Much had been learned about the placement of ribosomal proteins within those shapes. The secondary structures of the ribosomal RNAs (rRNAs) had been worked out, and sites on rRNAs where ribosomal proteins bind had been identified. In addition, the structures of several ribosomal proteins and a few rRNA fragments were known at atomic resolution in isolation. However, no one was so deluded as to imagine that Journal of Biology 2009, 8:8 8.2 Journal of Biology 2009, (a) Volume 8, Article 8 Moore http://jbiol.com/content/8/1/8 (for example [4,5]), and by 1988 the technology needed for ribosome crystallography was falling into place. L11 arm Small Subunit L1 arm head body Large Subunit (b) Figure 1 The ribosome at low resolution. The images shown here are photographs of plaster models of the two ribosomal subunits made by James Lake. They were derived from his EM images of the two ribosomal subunits from E. coli [45]. The resolution is about 40 Å. (a) The large subunit (left) and the small subunit (right) with some of their landmarks indicated. (b) The arrangement of the two subunits in the complete ribosome. structural information of this sort would ever explain ribosome function. The only two approaches for addressing the need for structural information that seemed promising in the 1980s were X-ray crystallography and electron microscopy. The first ribosome crystals, reported by Yonath, Wittmann and colleagues in 1980 [2], diffracted poorly; but, as the years went by, crystals were obtained of ribosomes and ribosomal subunits from many prokaryotic species (for example [3]), and the resolutions of the diffraction patterns of the best of them improved. The unit cells of ribosome crystals are very large and, consequently, ribosome crystals diffract X-rays so weakly that useful data can be collected from them only at synchrotron light sources. In 1980 the technology for doing macromolecular crystallography at synchrotrons was primitive, but major advances were made in the 1980s and thereafter Electron microscopy seemed promising because methods were being developed for obtaining three-dimensional electron density maps of biological objects from their twodimensional electron microscopic (EM) images [6]. Although the theory of image reconstruction is simple, its application to objects like the ribosome, for which the images to be reconstructed are those of isolated, randomly oriented particles, was fraught with difficulties. Nevertheless, by 1988 it seemed likely that ribosome reconstructions would eventually emerge with resolutions high enough to allow tRNAs to be visualized bound to the ribosome. Once that threshold was crossed, it seemed to me that EM would start contributing to our understanding of protein synthesis. My optimism notwithstanding, nothing published between 1988 and 1995 would have led the unbiased observer to conclude that the ribosome was likely to return any time soon. The advances made in EM reconstructions did not seem dramatic, and the papers published on ribosome crystallography were records of frustration. Ribosome crystallography had run aground on the shoals of the classic problem in macromolecular crystallography, the so-called phase problem, and it was unclear if it would ever get unstuck. Crystal structures are three-dimensional models of molecules that are generated by fitting chemical structure into experimentally determined, three-dimensional maps that display the distributions of electrons in those molecules. Electron density maps can be computed from crystal diffraction data only if the phases associated with each of the tens of thousands of reflections in such datasets are known. If there is no prior knowledge about the threedimensional structure of a macromolecule, phases must be measured experimentally. In the end, the experimental technique that contributed the most to solving ribosome crystal structures was the heavy atom multiple isomorphous replacement (MIR) method that Perutz devised in the 1950s to solve the structure of hemoglobin. (...truncated)