Structural Basis for the Allosteric Mechanisms Regulating Ribosome Function

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SUMMARY The ribosome is a complex molecular machine responsible for decoding the mRNA and producing all proteins in every organism. The process entails the selection of tRNAs, peptide bond formation, tRNA movement by one codon each elongation cycle, and release of the polypeptide chain. Translation factors are key regulators of ribosome function, modulating the conformation of the ribosome itself and of tRNAs. Our knowledge of ribosome functioning has benefited immensely from structural approaches that elucidated mechanisms of translation elongation and stop codon recognition during termination at a molecular level. Because the ribosome is the target for most of the clinically useful antibiotics, many structures of the ribosome in complex with the factors and inhibitors have allowed development of superior antibiotics. Remarkably, however, the mechanisms for two of the most important steps of protein synthesis, initiation and ribosome recycling, have remained unclear. Translation initiation and recycling of the ribosome into subunits mark the beginning and the end of the protein synthesis cycle, and therefore a better understanding of the molecular aspects of these processes could open the door to new therapeutics. Our recent findings reveal an unsuspected similarity between translation initiation and ribosome recycling: in both steps, the tRNA in the peptidyl (P) site adopts a highly similar conformation that is induced by translation factors. Despite this, the fate of the codon-anticodon interaction must be different because during translation initiation, the start codon is recognized by the initiator tRNA and during recycling, the codon-anticodon base pairing in the P site is expected to be disrupted. This suggests that the state of base pairing between the mRNA and the P-site tRNA is a major control element of ribosome functioning, an aspect of translation that has been so far overlooked. To gain insights into the molecular mechanisms of ribosome recycling and translation initiation, we propose to study unconventional aspects of translation. Hence, in Aim 1, we will determine the molecular mechanism of ribosome recycling in the human pathogen Pseudomonas aeruginosa that is facilitated by the unorthodox elongation factor G-1A (EF-G1A), a specialized EF-G that exclusively functions in ribosome recycling. In Aim 2, we will determine how initiation factor 2 (IF2) in P. aeruginosa recognizes the initiator tRNA independently of the formylation state of the methionine residue. In Aim 3, we will characterize how a codon-anticodon mispair with the initiator tRNA in the P site allosterically triggers a “quality check” by the ribosome that alters the decoding properties of the aminoacyl (A) site. These aims will be accomplished using multidisciplinary approaches, including state-of-the-art cryo-electron microscopy (cryo- EM) and X-ray crystallography of large functional ribosome complexes, together with biochemical methods such as stopped flow kinetic experiments and ribosome binding assays. The anticipated findings will fill important gaps in knowledge of ribosome functioning and may offer unsuspected opportunities for structure-guided development of new inhibitors of protein synthesis.