Poster abstracts
Poster number 166 submitted by Xingchen Liu
TruB-mediated tRNA pseudouridylation modulates translation during stress in E.coli
Xingchen Liu (University of Michgan, Department of Chemistry), Kristin S. Koutmou (University of Michgan, Department of Chemistry)
Abstract:
Pseudouridine (Ψ) is an abundant and evolutionarily conserved RNA modification that contributes to RNA folding and stability. In Escherichia coli, TruB catalyzes formation of Ψ55 in tRNA, a modification that stabilizes tRNA structure and may influence its function during translation. However, whether TruB-mediated Ψ55 affects translation and protein expression during stress remains unclear. To address this question, we use translation inhibitors that act at distinct stages of translation, allowing us to identify which steps are influenced by TruB-mediated tRNA pseudouridylation. Spot-plating assays revealed that TruB-dependent stress responses vary with the mechanism of translation inhibition. Using a reconstituted in vitro E. coli translation system, we further found that loss of TruB altered both the kinetics and endpoint yield of tRNAPhe-dependent dipeptide formation in an inhibitor-specific manner. Ongoing tripeptide formation assays test how inhibitors that disrupt translocation affect the rate of third amino acid addition. Nanopore direct RNA sequencing will map stress-dependent changes in pseudouridylation and other modifications of TruB-targeted tRNAs, while reporter assays and quantitative proteomics will determine how these changes affect protein expression. Together, these findings support a model in which TruB-mediated tRNA pseudouridylation modulates translation in a stress-specific manner.
Keywords: Pseudouridine, tRNA modification, Translational stress