Functional Characterization Of Accessory Proteins And Novel Activities In Direct And Indirect Trna Aminoacylation

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