From the Structure of the Ribosome to the Design of New Antibiotics Abstract Structural studies of the ribosome exemplify the evolution of structural studies in cell biology from the early negatively stained images of macromolecular assemblies in whole cells, to a detailed atomic understanding of the mechanism of action of a large assembly. The earliest electron microscopic (EM) images by George Palade capturing the ribosome in the cell were initially called Palade Particles. Biochemical studies in the 60’s showed that the larger subunit of this 2.6 MDal RNA-protein assembly catalyzed peptide bond formation while interactions of the anticodon of tRNA with mRNA bound to the small subunit effected translation of the message; the binding of the aminoacyl-tRNA to the A site and binding of the peptidyl-tRNA to the P site were identified, and translocation of the peptidyl-tRNA from the A site to the P site following peptide bond formation was hypothesized. Proceeding from the early reconstructions of the shapes of the two interacting subunits from negatively stained images by Jim Lake (1976) to the current atomic resolution structures of the 70S ribosome and of its large and small subunits captured in various functional states, the mechanistic level of structural insights into ribosome function now exceeds that achieved in the early structural studies of lysozyme, carboxypeptidase and ribonuclease. Mechanistic details of the decoding of messenger RNA at the atomic level have been derived by the Ramakrishnan lab from 3.0 Å resolution structures of the 30S ribosomal subunit complexed with mRNA and tRNA substrate fragments as well as more recent structures of tRNA substrates or fragments complexed with the 70S ribosome determined at resolutions between 3.7 Å and 2.8 Å by the Noller and Ramakrishnan labs. Structural insights into the peptidyl transferase reaction, as well as its inhibition by antibiotics, have come from structures of substrate and intermediate complexes with the 50S ribosomal subunit at resolutions that range variously between 3.3 Å and 2.3 Å from the Steitz lab. The first atomic model of the 70S ribosome derived from a 5.5 Å resolution map by the Noller lab using the atomic structures of the 30S and 50S subunits showed the interactions between the two subunits and the general positions of tRNA molecules bound to the A-, P- and E-sites, while the most recent higher resolution structures of the 70S ribosome show further details of the interactions made by tRNAs with the P site and E site. Also, a more complete and detailed structure of the ligand free 70S E. coli ribosome from the Cate lab has shown two conformations of the “head” domain of the small subunit that is related to the process of tRNA and mRNA translocation. Structures of the release factors 1 and 2 and the appropriate mRNAs bound to the 70S ribosome calculated from the Noller and Ramakrishnan labs provide insights into the termination of polypeptide synthesis. Recent crystal structures of the 70S ribosome captured in various states of tRNA delivery by elongation factor Tu and translocation by elongation factor G from the Ramakrishnan lab have shown EF-Tu delivering an aminoacyl-tRNA and EF-G promoting translocation as well as rachet-like relative rotations of the large and small subunits. More recently, we have also obtained structures of the 70S ribosome with protein factors. The protein factor EFP stimulates the formation of the first peptide bond, and our structure of the 70S ribosome with f-met-tRNA and EFP bound shows that the EFP binds adjacent to the P-site tRNA interacting with both the anticodon stem-loop and the acceptor stem. It is presumably positioning the P-site tRNA for peptide bond formation. We have also determined the structures of the 70S ribosome with two different hibernation factors. One binds the A site on the 30S subunit that overlaps with the P-site and A-site tRNAs, thereby preventing their binding. The other factor binds near the 3′ end of the 16S rRNA where the Shine-Delgano m-RNA sequence binds, which would also prevent the initiation of protein synthesis. These structural studies of the ribosome are not only providing a detailed look at the process of protein synthesis, but also demonstrate that the ribosome a ribozyme and that rRNA undergoes substrate ligand induced conformational changes in order to achieve specificity, just as is seen in protein enzymes. The ribosome is 2/3 RNA and 1/3 protein. Our structural studies of the Haloarcula marismortui (Hma) large subunit showed that the site of peptide bond formation (the peptidyl transferase center) consists entirely of rRNA and our structures of many different complexes of the large subunit with various substrate, intermediate and product analogues, along with kinetic and biochemical studies of others, have illuminated the mechanism of peptide bond formation. Binding of the correct substrate to the A site results in a conformational change in the rRNA and a reorientation of the peptidyl group of the P-site substrate. The 2’OH of A76 of the A-site tRNA is H-bonded to the -amino group of the A-site substrate. This structure of the pre-reaction substrate complex and other kinetic and biochemical data support a proton shuttle mechanism in which the 2’OH of A76 receives a proton from the attacking -amino group thereby enhancing its nucleophilicity, while it donates a proton to the 3′ oxygen of A76 of the peptidyl-tRNA as it is being deacylated. A movie of this process (with music) based on many structures will show the mechanism of peptide bond formation. The ribosome is a major target of antibiotics which are seen to bind either the large or the small subunit or to both subunits simultaneously. We have determined the structures of many different families of antibiotics bound to the Hma large subunit or to the Thermus thermophilus (T.th.) 70S ribosome, and those structures inform how the various antibiotics inhibit protein synthesis by the ribosome. They also suggest why various resistance mutations in the ribosome make the ribosome insensitive to specific antibiotics. We have also determined the structures of complexes of mutant ribosomes with antibiotics to validate the proposed mechanisms of antibiotic resistance produced by the mutations. The rise of antibiotic resistant bacteria is becoming a major global health problem. MRSA is reported to now result in about 100,000 deaths annually world-wide. The structures of our antibiotic complexes with the ribosome are currently being used for structure based drug design to create new compounds that are effective against MRSA and other antibiotic resistant bacterial strains. The strategy being employed by Rib-X Pharmaceuticals, Inc., in New Haven, Connecticut is to design new compounds that chemically link a portion of one known antibiotic to a part of another antibiotic that is observed to bind to an adjacent site. Using computational approaches for the design of new compounds, many cycles of compound synthesis and evaluation are resulting in new potential antibiotics that are effective against resistant strains are being created. One compound made by Rib-X Pharmaceuticals, Radezolid, has successfully completed phase II clinical trials and many other compounds are in their antibiotic development pipeline. Our structural studies of the ribosome and its complexes with many functionally important ligands are not only providing important insights into how this macromolecular machine works, but are now also leading to practical benefits to human health. References: Moore, P.B. and Steitz, T.A. The roles of RNA in the synthesis of protein. In: RNA World, 3rd edition, (R.F. Gesteland, T.R. Cech and J.F. Atkins, eds.), Cold Spring Harbor Laboratory Press, pp. 257-285 (2006). Steitz. T.A. A structural understanding of the dynamic ribosome machine. Nature Reviews: Mol. Cell Biol. 9: 243-253 (2008). Steitz, T.A. From the structure and function of the ribosome to new antibiotics. Angew. Chem. Int. Ed. (Nobel Lecture) 49: 4381-4398 (2010). Schmeing, T.M. and Ramakrishnan, V. What recent ribosome structures have revealed about the mechanism of translation. Nature 46: 1234-1242 (2009).