Substrate Divergence in Aminoacyl-tRNA Biosynthesis

描述(由申请人提供)： 氨酰-tRNA的生物合成是准确翻译遗传密码的基础。并不是所有的氨基酰-tRNA合成酶(生物合成氨基酰-tRNA的酶)都存在于所有生物体中，这引发了关于遗传密码翻译和进化的准确性的重要问题。这项建议集中在人类致病细菌幽门螺杆菌中谷氨酰tRNA(Gln)的生物合成。幽门螺杆菌缺少编码谷氨酰基和天冬酰胺基tRNA合成酶的基因(分别为GlnRS和AsnRS)。因此，Gln-tRNA(Gln)和Asn-tRNA(Asn)是通过错酰化的tRNA中间体[Glu-tRNA(Glu)和Asp-tRNA(Asn)]生物合成的。这些AA-tRNA由依赖于Gin的氨基转移酶Glu-ADT进行氨基酸修复。幽门螺杆菌基因组还编码两种谷氨酰tRNA合成酶(GluRS1和GluRS2)。GluRS2特别有趣，因为它已经失去了对其“同源”tRNA(Glu)底物进行氨基酰化的能力，只能生物合成Glu-tRNA(Gln)。 准确的tRNA氨酰化对细菌的生存是绝对重要的，但很明显，不同的细菌(特别是幽门螺杆菌)已经适应了不同的tRNA氨酰化机制。这些不同的机制是如何促进细菌健康的尚不清楚。这项建议的主要主题是表征和测试幽门螺杆菌在体外和体内使用的准确性机制。这些实验将提供对细菌适应和蛋白质翻译分歧的洞察。该提案分为三个目标。第一部分将描述GluRS2独特tRNA特异性背后的分子机制。第二项研究将研究幽门螺杆菌和大肠杆菌体内tRNA的直接氨酰化和间接氨酰化。第三个目的将检测Glu-ADT的tRNA选择性，以开始阐明该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.