Aminoacyl-tRNA biosynthesis is fundamental to accurate translation of the genetic code. Not all aminoacyl-tRNA synthetases (the enzymes that biosynthesize the aminoacyl-tRNAs) exist in all organisms, raising important questions about accuracy in translation and evolution of the genetic code. This proposal focuses on glutaminyl-tRNA(Gln) biosynthesis in the human pathogenic bacterium Helicobacter pylori. H. pylori is missing the genes encoding glutaminyl- and asparaginyl-tRNA synthetases (GlnRS and AsnRS, respectively). Consequently, Gln-tRNA(Gln) and Asn-tRNA(Asn) are biosynthesized via misacylated tRNA intermediates [Glu-tRNA(Glu) and Asp-tRNA(Asn), respectively]. These AA-tRNAs are amidatively repaired by a Gin-dependent amidotransferase, Glu-Adt. The H. pylori genome also encodes two glutamyltRNA synthetase (GluRSl and GluRS2). GluRS2 is particularly interesting because it has lost the ability to aminoacylate its """"""""cognate"""""""" tRNA(Glu) substrates and only biosynthesizes Glu-tRNA(Gln). The importance of accurate tRNA aminoacylation is absolute to bacterial viability and yet it is clear that different bacteria (H. pylori in particular) have adapted to use divergent tRNA aminoacylation mechanisms. How these different mechanisms contribute to bacterial fitness remains unclear. The major theme of this proposal is to characterize and test the accuracy mechanisms used by H. pylori in vitro and in vivo. These experiments will provide insight into bacterial adaptation and divergence in protein translation. The proposal is divided into three aims. The first will characterize the molecular mechanisms behind the unique tRNA specificity of GluRS2. The second will examine direct versus indirect tRNA aminoacylation in vivo in both H. pylori and in E. coli.
The third aim will examine the tRNA selectivity of Glu-Adt, to begin to elucidate the mechanisms by which this enzyme selects its two distinct AA-tRNA substrates.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Fabian, Miles
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Johns Hopkins University
Schools of Arts and Sciences
United States
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