All life requires the active participation of a class of RNA molecules known as transfer RNA (tRNA) and many biological processes act to ensure the proper functioning of this tRNA pool by removing damaged or defective tRNA molecules before they can cause harm to the cell. This project is focused on discovering new pathways for protecting bacterial cells from damaged tRNA, as might occur occasionally due to mistakes made by the enzymes that are responsible for producing tRNA, or due to growth in stressful conditions. Although some of these pathways have been identified in eukaryotes, the fate of these damaged tRNAs is not completely understood in Bacteria. In addition, the research will determine the function of potentially novel tRNA metabolism enzymes that were identified by integrative data mining and appear likely to play important but unknown roles in tRNA metabolism. The project will train one graduate student and two post-doctoral fellows to use cutting-edge multidisciplinary approaches to solve fundamental biological problems, and will develop three classroom-ready online modules using biological data-mining tools for high school teachers.
The goal of this research is to discover novel regulatory processes involving tRNA turnover in prokaryotes and to understand the role of tRNA modifications in these processes. This interdisciplinary project integrates computational analyses with innovative technologies that encompass bacterial genetics, RNA biochemistry, bioanalytical chemistry and genomics and the use of novel systems-level analysis tools to survey the modification and cleavage profiles of all tRNAs in the cell. The research will analyze the role of modifications as determinants for tRNA integrity in Bacteria by conducting screens for synthetic growth phenotypes, combining deletions in tRNA modification genes under different stress conditions and measuring tRNA levels, modification status and fragment population in strains lacking specific modifications. In addition, preliminary comparative genomic analysis has linked bacterial enzymes related to tRNA metabolism, but whose precise biological function is unknown, to both tRNA modification and stress resistance, suggesting they participate in novel mechanisms involving tRNA degradation and/or repair. This hypothesis will be tested by analyzing tRNA pro-files in E. coli derivatives lacking the corresponding genes and by identifying specific RNA substrates for these orphan RNA metabolism enzymes.
