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Functional and mechanistic characterization of two tRNA modifying enzymes
(Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2016) Keffer-Wilkes, Laura C.; University of Lethbridge. Faculty of Arts and Science; Wieden-Kothe, Ute
The formation of pseudouridine and 5-methyluridine (m5U) in the T-arm of transfer RNAs (tRNAs) is near-universally conserved. These two modifications are formed in Escherichia coli by the pseudouridine synthase TruB and the S-adenosylmethionine-dependent methyltransferase TrmA, respectively. In this thesis, I investigate the function and mechanisms of these two tRNA modifying enzymes. First, in vitro and in vivo analysis of TruB reveals that this enzyme is acting as a tRNA chaperone which proves a long outstanding hypothesis. Secondly, characterization of ligand binding by TrmA shows that binding is cooperative and disruption of tRNA elbow region tertiary interactions by TrmA is essential for efficient tRNA binding and catalysis, leading to future analysis. In conclusion, my studies further our understanding of the mechanism and function of tRNA modifications and modifying enzymes, as well as shed light on why all cells invest substantial resources into fine-tuning the chemical composition of tRNAs.