Home WakeSpace Scholarship › Electronic Theses and Dissertations

BIOSYNTHESIS, FUNCTION, AND CHARACTERIZATION OF BACILLUS SUBTILIS 2-THIOURIDINE TRNA

Electronic Theses and Dissertations

Item Files

Item Details

title
BIOSYNTHESIS, FUNCTION, AND CHARACTERIZATION OF BACILLUS SUBTILIS 2-THIOURIDINE TRNA
author
Edwards, Ashley MaryAnn
abstract
Transfer RNAs (tRNAs) are key components in protein synthesis, promoting the translation of the genetic code. In addition, tRNA performs non-canonical roles during cellular response to environmental changes and stressors, charging tRNA with increasingly complex, regulatory roles during metabolic homeostasis. Thio-modifications to tRNA are essential for translational fidelity as well as epitranscriptomic regulation and cellular response to metabolite fluctuations in the cell. More specifically, the 2-thiouridine tRNA modification occurs at U34 of tRNALys,Glu,Gln and is indispensable for codon-anticodon recognition, translational accuracy, and frameshift prevention. In the Gram-positive organism Bacillus subtilis, the cysteine desulfurase YrvO directly passes sulfur to its dedicated acceptor protein, thiouridylase MnmA, for the synthesis of 2-thiouridine (s2U). This pathway exploits a distinct sulfur trafficking route when compared to the parallel orthologous pathway involving the Escherichia coli promiscuous cysteine desulfurase, IscS, for s2U synthesis. This dissertation describes the development and application of analytical methods for analyzing pathway metabolites that were used in tandem with previously established mechanistic, biochemical, and structural approaches. Using this experimental framework, this study established that s2U responds to sulfur availability in B. subtilis. That is, nutrient sulfur-limiting conditions affect the levels of s2U biosynthetic components, YrvO and MnmA, and consequently impact the relative abundance of s2U derivatives mmn5s2U and cmnm5s2U. Under these conditions, tRNALys,Glu,Gln substrates are responsive to cellular sulfur availability, providing an additional role for 2-thiouridine as a sensor of sulfur availability in Bacillus subtilis. Herein, we also highlight that YrvO and MnmA possess highly specific structural features that dictate their sulfur trafficking flow, as well as the types of substrates they can accept. We present the first report of B. subtilis MnmA Fe-S cluster coordination in vitro and provide evidence that cluster-bound MnmA exhibits enhanced activity for U34 thiolation when compared to the apo form of this enzyme. Taken together, the work outlined in this dissertation expands the roles of S-tRNA modifications and gives insight into the conserved and distinct features of their associated pathways across bacterial species. Findings from this dissertation will guide future works linking the roles ascribed to modified tRNAs and their biosynthetic components to factors influencing their regulation and activities.
subject
Bacillus subtilis
LCMS
SAR
Sulfide
Sulfur
tRNA
contributor
Dos Santos, Patricia C (advisor)
Tracy, Chris M (committee member)
Alexander, Rebecca W (committee member)
Stich, Troy A (committee member)
King, Stephen B (committee member)
date
2023-01-24T09:35:41Z (accessioned)
2022 (issued)
degree
Chemistry (discipline)
embargo
2027-12-31 (terms)
2027-12-31 (liftdate)
identifier
http://hdl.handle.net/10339/101770 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

Usage Statistics