Substrate Divergence in Aminoacyl-tRNA Biosynthesis

氨酰基-tRNA 生物合成中的底物差异

• 批准号: 7367941
• 负责人: TAMARA L HENDRICKSON
• 金额: $ 24.11万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367941
• 关键词: Address; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacyl-tRNA Biosynthesis Pathway; Aminoacylation; Anabolism; Anti-Bacterial Agents; Archaea; Asparagine-Specific tRNA; Asparagine-tRNA ligase; Aspartate-tRNA Ligase; Aspartic Acid-Specific tRNA; Bacteria; Bacterial Genome; Categories; Complex; Condition; Cytoplasm; Discrimination; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Evolution; Foundations; Genes; Genetic Code; Genome; Genomics; Glutamate-tRNA Ligase; Glutamic Acid-Specific tRNA; Glutamine; Glutamine-Specific tRNA; Glutamine-tRNA ligase; Goals; Helicobacter pylori; Human; In Vitro; LGLA; Ligase; Maps; Molecular; Organism; Pathogenicity; Pattern; Prokaryotic Cells; Proteins; Pylorus; Research Personnel; Role; Running; Saccharomyces cerevisiae; Specificity; Testing; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; Work; aspartic acid-tRNA; fascinate; fitness; glutamic acid-tRNA; glutamine-tRNA; glutamyl-tRNA(Gln); improved; in vivo; insight; interest; mutant; novel; pathogen; pathogenic bacteria; programs; repair enzyme; repaired; research study

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.

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。并非所有的氨酰基-tRNA 合成酶（生物合成氨酰基-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准确性的重要问题。该提案重点关注人类致病菌幽门螺杆菌中谷氨酰胺酰-tRNA（Gln）的生物合成。幽门螺杆菌缺失编码谷氨酰胺酰和天冬酰胺酰 tRNA 合成酶（分别为 GlnRS 和 AsnRS）的基因。因此，Gln-tRNA(Gln) 和 Asn-tRNA(Asn) 是通过异酰基化 tRNA 中间体 [分别是 Glu-tRNA(Glu) 和 Asp-tRNA(Asn)] 生物合成的。这些 AA-tRNA 由 Gln 依赖性酰胺转移酶 Glu-Adt 进行酰胺修复。幽门螺杆菌基因组还编码两种谷氨酰RNA合成酶(GluRS1和GluRS2)。 GluRS2 特别有趣，因为它失去了氨酰化其“同源”tRNA (Glu) 底物的能力，并且仅生物合成 Glu-tRNA (Gln)。 准确的 tRNA 氨酰化对于细菌的生存能力是绝对重要的，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。这些不同的机制如何促进细菌健康仍不清楚。该提案的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。这些实验将深入了解细菌的适应和蛋白质翻译的差异。该提案分为三个目标。第一个部分将描述 GluRS2 独特 tRNA 特异性背后的分子机制。第二个将检查幽门螺杆菌和大肠杆菌体内的直接与间接 tRNA 氨酰化。第三个目标是检查 Glu-Adt 的 tRNA 选择性，以开始阐明该酶选择其两种不同 AA-tRNA 底物的机制。