Substrate Divergence in Aminoacyl-tRNA Biosynthesis

描述（由申请人提供）： 氨酰-tRNA生物合成是遗传密码准确翻译的基础。并非所有的氨酰-tRNA合成酶（生物合成氨酰-tRNA的酶）存在于所有生物体中，这就提出了关于遗传密码翻译和进化准确性的重要问题。本提案的重点是人致病性细菌幽门螺杆菌中的谷氨酰-tRNA（Gln）生物合成。H.幽门螺杆菌缺失编码氨酰-和天冬酰胺酰-tRNA合成酶（分别为GlnRS和AsnRS）的基因。因此，Gln-tRNA（Gln）和Asn-tRNA（Asn）通过错酰化的tRNA中间体[分别为Glu-tRNA（Glu）和Asp-tRNA（Asn）]生物合成。这些AA-tRNA通过Gin依赖性酰胺转移酶Glu-Adt进行酰胺修复。螺杆pylori基因组还编码两种谷氨酰tRNA合成酶（GluRS 1和GluRS 2）。GluRS 2是特别有趣的，因为它已经失去了氨基酰化其“同源”tRNA（Glu）底物的能力，并且仅生物合成Glu-tRNA（Gln）。 准确的tRNA氨酰化对细菌的生存力是绝对重要的，但很明显，不同的细菌（H。pylori）已经适应使用不同的tRNA氨酰化机制。这些不同的机制如何促进细菌适应性仍不清楚。本提案的主要主题是表征和测试H. pylori的体外和体内研究。这些实验将提供深入了解细菌的适应和蛋白质翻译的分歧。该提案分为三个目标。第一个将表征GluRS 2独特的tRNA特异性背后的分子机制。第二个实验将在两种H. pylori和E.杆菌第三个目标是研究Glu-Adt的tRNA选择性，开始阐明这种酶选择其两种不同的AA-tRNA底物的机制。

DESCRIPTION (provided by applicant): 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.