Structural bases for protein synthesis quality control - PROJECT SUMMARY Accurate and faithful ribosome-directed protein synthesis is an essential step of gene expression. During this process, the codons in mRNAs are systematically decoded by tRNAs with the assistance of translation protein factors. The bacterial ribosome is the target for the majority of antibiotics used in hospital settings and therefore, understanding how the ribosome monitors the quality and accuracy of synthesized proteins could provide unanticipated opportunities for the development of new and improved therapeutics. Whereas the function of canonical translation factors has been studied for decades, the mechanisms by which the same protein factors control the quality of protein synthesis are enigmatic. This represents an aspect of translation regulation that has remained largely unexplored. Several of the quality control steps are mediated by canonical translation factors, such as release factor 3 (RF3) and initiation factor 2 (IF2). RF3 is known to recycle translation termination factors RF1 and RF2 from the ribosome; however, RF3 is also implicated in the release of the nascent peptide chain from mismatched ribosome complexes, in peptidyl-tRNA drop-off, and co-translational protein folding. Similarly, besides ensuring accurate and timely initiation of protein synthesis, IF2, together with initiation factor 1 (IF1), can monitor translation by stimulating peptidyl-tRNA drop-off, suggesting that IF1 and IF2 can function on elongating ribosomes. My group recently elucidated the mechanism of GDP exchange in RF3 catalyzed by the ribosome termination complex, illustrating how the ribosome can function as the guanine nucleotide exchange factor (GEF) for RF3. Our cryo-EM structures of the 70S ribosome simultaneously bound to RF3 and RF1, and to IF1 and IF2, provide a solid foundation for us moving forward to investigate fundamental aspects of translation quality control in bacteria mediated by RF3, as well as by IF1 and IF2. We further propose to elucidate how non- canonical translation factors, such as elongation factor 4 (EF-4) and ATP-binding cassette family (ABCF) proteins, modulate the conformation of the P-site tRNA and of the attached nascent peptide chain to rescue translationally compromised ribosomes and confer antibiotic resistance. Altogether, the proposed studies will provide mechanistic insights into the function of canonical and non-canonical translation factors in protein synthesis quality control and rescue of stalled ribosomes, and in the long-term may offer unsuspected therapeutic opportunities for the treatment of bacterial infections.
