Access Type

Dissertation/Thesis

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Tamara L. Hendrickson

Abstract

TRNA AMINOACYLATION: NEW PROTEIN PLAYERS AND NEW REACTIONS

by

WHITNEY N. WOOD

May 2019

Advisor: Dr. Tamara L. Hendrickson

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Protein translation must usually occur with high accuracy for an organism to survive. However, Helicobacter pylori, Staphylococcus aureus, and many other microorganisms including important human pathogens, lack one or more aminoacyl-tRNA synthetase (aaRS), the enzymes that typically aminoacylate tRNAs for ribosomal translation. These organisms must use an indirect pathway to aminoacylate some tRNAs. Specifically, H. pylori lacks the genes that encode for asparaginyl- and glutaminyl-tRNA synthetases (AsnRS and GlnRS, respectively). Instead, H. pylori uses a two-step, indirect pathway to synthesize Asn-tRNAAsn and Gln-tRNAGln. This process requires two misacylating tRNA synthetases (a non-discriminating aspartyl-tRNA synthetase, ND-AspRS, and a misacylating glutamyl-tRNA synthetase 2, GluRS2) and a glutamine-dependent amidotransferase called GatCAB. We have recently discovered a protein called Hp0100 that promotes formation of a stable complex between ND-AspRS, Hp0100, and GatCAB called the Asn-transamidosome. Hp0100 enhances GatCAB’s ability to produce both Asn-tRNAAsn and Gln-tRNAGln and it hydrolyzes ATP in the presence of either Asp-tRNAAsn or Glu-tRNAGln, the precursors to Asn-tRNAAsn and Gln-tRNAGln, respectively. (Red text indicates misacylation.) This dissertation focuses on three different research projects. Chapter 2 describes our evaluation of a shortened ortholog of Hp0100 from S. aureus. Full-length orthologs of Hp0100 are restricted to the ε-proteobacteria; however, we have recently discovered truncated orthologs comprising the N-terminal 2/3 of Hp0100 outside of this clade. The truncated ortholog from S. aureus (Sa2591) is compared to Hp0100 to assess if it is a functional ortholog of Hp0100. The predicated model structure of Sa2591 showed several conserved cysteine residues clustered together with a CXXC motif next to one of the ATPase active sites. Chapter 3 focuses on our characterization of this putative metal-binding site and Sa2591’s potential involvement in the synthesis of a tRNA modification. Chapter 4 reports our discovery that some aaRSs hydrolyze ATP to ADP in the presence of tRNA. All aminoacyl-tRNA synthetases (aaRSs) catalyze the ATP-dependent aminoacylation of tRNAs, producing AMP and PPi as side products. We are unaware of any evidence that aaRSs can produce ADP instead of AMP. We characterized this activity in 25 different aaRSs and propose a function for ADP formation.

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