Cellular and molecular functions of transfer RNA modifying enzymes
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Schultz, Sarah Kai-Leigh
University of Lethbridge. Faculty of Arts and Science
Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
All transfer RNAs (tRNAs) are highly modified. Several modifications are found within the tRNA elbow, including the universally conserved 5-methyluridine (m5U) 54 and pseudouridine (Ψ) 55 modifications in the T arm and 7-methylguanosine (m7G) 46 in the tRNA variable loop, formed by the bacterial tRNA modifying enzymes TrmA, TruB, and TrmB, respectively. Despite conservation of these enzymes throughout all domains of life, they are not essential for bacterial growth in ideal conditions. The overall goals of this thesis were to investigate molecular determinants for tRNA modification by these enzymes in vitro and determine cellular roles for these modifications in vivo. Specific chapters address (I) the preferential tRNA modification status for modification by TrmA, TruB, and TrmB, (II) the mechanism of tRNA binding by TrmB, and (III) the cellular roles for TrmA and TruB. Taken together, these studies reveal the mechanistic basis for TrmA and TruB acting in the early stages of tRNA maturation and provide justification for using an unmodified tRNA substrate for the rapid-kinetic analysis of TrmB binding tRNA, which reveals that prior S-adenosylmethionine binding is necessary for stable and rapid tRNA binding by TrmB. Additionally, residues distant from the active site within TrmB were identified to contribute for tRNA binding. Finally, tRNA sequencing, proteomics, and biochemical studies uncover that TrmA and TruB fine-tune global tRNA function by enhancing further tRNA modification, aminoacylation, and protein translation. In summary, this thesis provides insight into the tRNA binding and modification activity of three highly conserved tRNA modifying enzymes and identifies cellular roles of TrmA and TruB for tRNA modification, folding and protein synthesis, thus explaining why these enzymes are so highly conserved. This research sets the stage for studying the mechanisms and functions of further tRNA modifying enzymes, in addition to modification enzymes that target other species of RNAs.
transfer RNAs , tRNA modifying enzymes , TrmA , TruB , TrmB , molecular determinants , tRNA modification