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Date of Award
Tamara L. Hendrickson
Indirect tRNA aminoacylation is essential for most bacteria and archaea, particularly when these species do not have genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS). In the absence of AsnRS, the first step in Asn-tRNAAsn synthesis involves misacylation of tRNAAsn with aspartate to produce Asp-tRNAAsn; this reaction is catalyzed by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Subsequently, in bacteria, an amidotransferase called GatCAB converts Asp-tRNAAsn to Asn-tRNAAsn. An analogous, two-step processes exist to produce Gln-tRNAGln. In this case, a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) misacylates tRNAGln to produce Glu-tRNAGln, which is then converted to Gln-tRNAGln by GatCAB. The central hub of the indirect tRNA aminoacylation pathway is the formation of a macromolecular complex called the transamidosome.
In Helicobacter pylori, the pathogenic bacterium that causes stomach ulcers and gastric cancers, Asn-transamidosome formation requires a protein partner called Hp0100, to form a stable, tRNA-independent complex. Hp0100 accelerates the GatCAB-catalyzed transamidation of Asp-tRNAAsn into Asn-tRNAAsn ~35 fold and of Glu-tRNAGln into Gln-tRNAGln ~3 fold. Our preliminary evidence suggests that Hp0100 contains two mutually exclusive ATP hydrolase domains, which are activated by Glu-tRNAGln and Asp-tRNAAsn, respectively.
This dissertation work focusses on four different but connected projects. Chapter 2 discusses the characterization of the Mycobacterium smegmatis Asn-transamidosome. Overexpression of GatCAB in M. smegmatis, a benign, close relative of Mycobacterium tuberculosis, and its purification resulted in the co-purification of ND-AspRS and several other proteins. These results represent the first successful purification of a Mycobacterial Asn-transamidosome from its native organism. Efforts to determine if ND-GluRS also co-purified with GatCAB were inconclusive. Two of the unknown proteins that reproducibly co-purify with GatCAB were identified as a universal stress protein (USP) and superoxide dismutase (SOD), which are stress-related proteins. Chapter 3 focuses on the discovery of an unexpected enzymatic activity of AspRSs. We discovered that some bacterial, discriminating AspRS (E. coli) and ND-AspRSs (H. pylori, M. smegmatis, and Staphylococcus aureus) are capable of aminoacylating E. coli tRNAGlu with glutamate to produce correctly aminoacylated Glu-tRNAGlu without glutamylating their “natural” tRNAs (tRNAAsp and tRNAAsn). Chapter 4 summarizes our efforts to optimize the overexpression and purification of Hp100 without a metal-binding tag. Finally, we have identified truncated orthologs of Hp0100 in several other pathogenic bacteria outside this clade. Chapter 5 reports our preliminary characterization of a truncated ortholog of Hp0100 called Sa2591 from S. aureus. Sa2591 shares some functional similarities with Hp0100 and may be a sensor for the presence of post-transcriptional modifications in tRNAAsn.
Rathnayake, Udumbara Menike, "Functional Characterization Of Accessory Proteins And Novel Activities In Direct And Indirect Trna Aminoacylation" (2019). Wayne State University Dissertations. 2244.