tRNA Synthesis Fidelity Mechanisms

tRNA 合成保真度机制

• 批准号: 7990800
• 负责人: SUSAN A MARTINIS
• 金额: $ 10.44万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990800
• 关键词: Acids; Active Sites; Address; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Antibiotics; Binding; Biochemical; Biochemistry; Cell Survival; Cells; Custom; Development; Engineering; Ensure; Enzymes; Escherichia coli; Event; Family; Funding; Future; Goals; Hydrolysis; Individual; Interdisciplinary Study; Investigation; Leucine; Life; Ligase; Link; Modeling; Molecular; Molecular Biology; Mutation; Organism; Pathway interactions; Pharmacologic Substance; Physiological; Process; Progress Reports; Protein Biosynthesis; Proteins; Research; Research Personnel; Ribosomes; Screening procedure; Site; Therapeutic; Transfer RNA; Transfer RNA Aminoacylation; Translations; X-Ray Crystallography; Yeasts; adenylate; base; computational chemistry; design; in vivo; interdisciplinary collaboration; leucine-tRNA; molecular site; mutant; novel; polypeptide; programs; tool

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases (aaRSs) comprise a family of twenty enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates ("pre-transfer" of arnino acid to tRNA) or misacylated aa- tRNA ("post-transfer"). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which complicates investigations to determine their respective molecular basis. E. coli leucyl-tRNA synthetase (LeuRS) is unique because it edits exclusively by a post-transfer mechanism. In the past funding cycle, the post-transfer editing activity was abolished and a pre-transfer editing pathway activated in E. coli LeuRS by a limited number of mutations. Thus, the E. coli wild-type and mutant LeuRS provide a powerful model to segregate the two aaRS fidelity mechanisms and characterize molecular determinants that are specific to pre- and/or post-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computational, biochemical, and molecular biology approaches to investigate translocation mechanisms for misactivated aminoacyl- adenylate intermediates in pre-transfer editing and mischarged tRNAs in post-transfer editing. It will also determine the physiological impact of the aaRSs on translational fidelity and cell viability. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.

描述（由申请人提供）：氨基酰基-TRNA合成酶（AARSS）包括一个对每个生物体必不可少的二十个酶的家族。每种酶都识别一个同源氨基酸，并将其连接到正确的tRNA。然后，“带电”的tRNA将核糖体处的氨基酸转移到生长的多肽链中。蛋白质合成的保真度完全取决于AARSS准确的底物识别。一些AARS已开发出编辑机制来纠正滥用的氨基酸。这些编辑的AARS通过水解两个底物溶解的氨基酰基 - 亚戊烯基（将arnino酸到tRNA的“前转移”）或降低的AA-tRNA（“后转移”）的水解清除了错误的氨基酸。尽管这些机制之一可能占主导地位，但编辑的大多数AARS似乎是通过转移前和转移后编辑的混合物进行的，这使研究复杂以确定其各自的分子基础。大肠杆菌Leucyl-tRNA合成酶（Leurs）是独一无二的，因为它仅由转移机制编辑。在过去的资金周期中，取消转移的编辑活动被废除，并通过有限的突变在大肠杆菌中激活了转移前的编辑途径。因此，大肠杆菌野生型和突变体Leurs提供了一个强大的模型，以隔离两种AARS保真机制并表征特定于转移后编辑的分子决定因素。该提案概述了一个跨学科研究计划，该计划结合了X射线晶体学，计算，生化和分子生物学方法，以研究在转移前编辑和误导后的TRNA中，在移动后编辑中，在转移前编辑和误导的TRNA中，用于误导的氨基酰胺中间体的易位机制。它还将确定AARS对翻译保真度和细胞生存能力的生理影响。对编辑机制的详细理解将使正在进行的药物研究受益于AARSS作为抗生素开发目标。它还将使AARSS重新设计以激活替代氨基酸以掺入定制设计的蛋白质中。这些新型蛋白质可以用作药物和技术应用中的治疗剂或重要工具。