Elucidating the role of peptidyl-tRNA hydrolase activities in translation regulation

Synthesis of proteins is a fundamental activity that sustains the life of all organisms. The process of synthesizing proteins inside living cells normally occurs efficiently and smoothly. However, occasionally, and even more so under stress conditions, the protein synthesis machinery encounters problems and stalls. Fortunately, cells are equipped with quality control systems dedicated to overcoming problems in protein synthesis. The multiple components and mechanisms of action of the quality control systems are far from being fully characterized and understood. In this project, the cellular factors and molecular mechanism of a previously unknown critical protein synthesis quality control system in bacteria will be studied. The resulting new knowledge will inform new strategies for combating bacterial infections. The research activities will be incorporated in educational programs to train students from high school, college, and graduate programs, as well as postdoctoral researchers. The research findings will be communicated to the scientific community through presentations at local, national, and international meetings and by publications in peer-reviewed journals. It has been assumed that the enzyme peptidyl-tRNA hydrolase (Pth) is essential for bacteria because of its ability to recycle transfer RNA (tRNA) molecules from peptide-tRNA complexes that dissociate from the ribosome during abortive cycles of translation. Recently, it has come to light that Pth is capable of cleaving peptidyl-tRNAs on ribosomes that have stopped making proteins with problematic sequences. This newly discovered involvement of Pth in translation quality control reveals that the reasons for the essentiality of this enzyme are yet to be fully understood. In this project, ribosome profiling (Ribo-Seq), an approach that reveals the positions of ribosomes genome-wide, will help identify the gene sites in two model bacteria (Escherichia coli and Bacillus subtilis) where Pth acts as a rescue factor for ribosomes struggling to translate specific sequences. In addition, biochemical and structural approaches will be used to reveal the mechanistic principles underlying Pth-driven ribosome rescue. The project will advance our understanding of strategies for protein synthesis quality control. The findings of the project may guide future research to design novel antibiotics and treatments for diseases associated with defective protein synthesis. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.