A systems-level view of the tRNA epitranscriptome : defining the role of tRNA abundance, stability, and modifications in the bacterial stress response

Abstract

In all organisms, the modulation of gene expression is a critical aspect of growth, development, and adaptation to environmental changes. Technological advancements in the post-genomic era have provided new 'omics tools for achieving a systems-level understanding of transcription and translation. This has led to an emerging appreciation for the complexity of post-transcriptional mechanisms regulating gene expression, including the pool of transfer RNA (tRNA) molecules within the cell and the spectrum of modified ribonucleotides that comprise the epitranscriptome. The studies presented in this thesis address both 'omic tool-building and a mechanistic understanding of the prokaryotic epitranscriptome. Observations that tRNA modifications and tRNA copy numbers change dynamically in response to environmental perturbations have led to the hypothesis that tRNA-mediated mechanisms contribute to the cellular stress response.

Members of the Mycobacterium tuberculosis complex provide a highly relevant model for investigating this mode of post-transcriptional regulation. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (Tb), one of the most prevalent infectious diseases in the world. During infection, Mtb is subjected to harsh conditions - including hypoxia, nutrient limitation, and macrophage-derived reactive oxygen/nitrogen species (ROS/RNS) - within avascular granulomas. Mtb has evolved to persist by dramatically remodeling its metabolism and entering a non-replicative, quasi-dormant state that renders it highly tolerant of host-inflicted immune assaults. This thesis investigates the mechanisms by which mycobacteria use a combination of tRNA modifications and tRNA copy number changes to orchestrate extensive remodeling of biochemical networks during starvation-induced persistence.

The technologies and questions applied to mycobacteria were also used to characterize the network of tRNA modifications and modifying enzymes in E coli, along with their role in tRNA surveillance and quality control. The results of our studies have led to new technologies with commercial potential and have advanced our understanding of the complex mechanisms governing gene expression.