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DISSECTING THE MECHANISM OF tRNAIle2-LYSIDINE SYNTHETASE AND THE EFFECTS OF OBSERVED EVOLVED MUTATIONS ON CATALYTIC CAPABILITIES

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title
DISSECTING THE MECHANISM OF tRNAIle2-LYSIDINE SYNTHETASE AND THE EFFECTS OF OBSERVED EVOLVED MUTATIONS ON CATALYTIC CAPABILITIES
author
Guinto, Ferdiemar Cardenas
abstract
Protein synthesis is critical in all forms of life. Errors in protein synthesis can result in misfolding events, protein degradation, or mRNA decay, which can ultimately lead to disease phenotypes. Certain tRNA modifications exist to prevent mischarging of the tRNA, mispairing at the ribosome, and misincorporation in the polypeptide chain. In most cases, tRNA modifying enzymes need only to be moderately specific as they act on multiple tRNA substrates in the cell. There are five primary tRNA modifying enzymes that perform chemistry at the wobble base of the tRNA anticodon. These five have been widely studied; most have solved crystal structures associated and modify multiple tRNA species from the total tRNA pool. However, one of these enzymes has only one cognate substrate and therefore needs to be much stricter than the others. tRNA isoleucine lysidine synthetase (TilS) is a bacterial exclusive wobble-base modifying enzyme that catalyzes the modification of cytidine at the wobble position of tRNAIle2CAU (C34) to the modified nucleoside lysidine (k2C), resulting in a tRNAIle2LAU (L34). This reaction is catalyzed by the formation of an adenylated intermediate at C34 (AMP-C34) and a subsequent attack by the ε-amino group of a lysine amino acid. The resulting lysidine modification is enough to prevent improper AUG pairing at the ribosome, restricting the modified tRNA to decode AUA codons.In most bacterial organisms, the TilS enzyme is necessary to decode the AUA isoleucine codon. Without this enzyme, it is possible that misincorporation would occur at these locations, leading to mistranslation events and resulting in improper protein synthesis. While the universal necessity of the enzyme is still in question, the tilS gene has been shown experimentally to be an essential bacterial gene in at least two organisms, Bacillus subtilis and Escherichia coli.1,2 Furthermore, a collaborative project between the Alexander lab at Wake Forest University and the Cooper lab from University of Pittsburgh School of Medicine has shown novel results stemming from evolutionary experiments with Burkholderia cenocepacia TilS (BcTilS). Through propagation of B. cenocepacia in nutrient-deficient media, a series of single nucleotide polymorphisms (SNPs) in the tilS gene arose. The Alexander lab demonstrated, in vitro and in vivo, that the resulting TilS mutations led to a notable decrease in lysidinylation activity. Interestingly, none of these mutations occurs in the active site of TilS. The reported biological importance of the tilS gene, the ramifications of the function and a potential knock-out phenotype, the enzyme’s unique mechanism and mode of action, and the observed mutations that arose in forced evolution experiments result in a unique system to investigate the properties of protein structure and function.
subject
Enzymes
Nucleic acids
Protein/RNA interactions
Proteins
contributor
Alexander, Rebecca W. (advisor)
Dos Santos, Patricia C. (committee member)
King, S. Bruce (committee member)
Stich, Troy A. (committee member)
date
2024-02-13T09:36:03Z (accessioned)
2023 (issued)
degree
Chemistry (discipline)
embargo
2026-02-12 (terms)
2026-02-12 (liftdate)
identifier
http://hdl.handle.net/10339/102899 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

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