A bioanalytical platform for interrogating the systems biology of tRNA modifications: Application to defining translational control mechanisms in bacterial stress responses

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Peter Dedon from the Massachusetts Institute of Technology to characterize a newly discovered, translation-based mechanism of bacterial stress response, in which stress causes reprogramming of dozens of tRNA modifications that regulate the translation of codon-biased transcripts to achieve selective expression of critical proteins. The studies will be initiated by comprehensively characterizing modified ribonucleosides in tRNA in Pseudomonas aeruginosa (PA) using a mass spectrometry-based platform developed in these labs. Following exposure of PA to hydrogen peroxide, changes in the spectrum of modified ribonucleosides will be quantified and modifications found to increase significantly under stress will be localized within specific tRNA molecules. Stress-altered wobble modifications will then be used to predict anticodon-codon interactions involved in selective translation of specific mRNAs for response and adaptation proteins, with proteomic validation of stress-induced changes in protein levels. Finally, a transposon mutant library will be used to define the biosynthetic pathways for stress-altered modifications by screening tRNA modification spectral changes in PA mutants selected by homology to other prokaryotes. The proposed studies will provide new insights into the translational mechanisms governing bacterial responses to environmental conditions, and the detailed molecular mechanisms that link translation to cell phenotype. The goal of these studies is to see if bacteria use a novel mechanism of cell survival and adaptation recently discovered in yeast and human cells. Using technology for quantifying the dozens of modified ribonucleotide building blocks of transfer RNA (tRNA) - the molecules controlling protein synthesis in all living organisms - it was discovered that stressed cells "reprogram" their tRNA modifications in a way that leads to selective production of proteins that are required to survive to the stress. The generality of this model will now be tested in the bacterium, Pseudomonas aeruginosa, to begin to understand how translational control mechanisms affect the ability of this important pathogenic microbe to survive and adapt during infection. The methods developed here will provide microbiologists with tools to systematically study RNA modifications in any type of bacterium, in principle, with potential broad impact on the basic science of cell and molecular biology. The studies will also provide students and scientists with specialized training in analytical technology and in a new fundamental mechanism of cell biology, while an outreach program will engage high school biology students with hands-on activities in cell culture, RNA isolation and mass spectrometry.