tRNA Synthetase Fidelity Mechanisms

tRNA 合成酶保真机制

基本信息

批准号：
8287046
负责人：
SUSAN A MARTINIS
金额：
$ 31.24万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8287046
关键词：
Active Sites Address Amino Acids Amino Acyl-tRNA Synthetases Aminoacylation Anti-Infective Agents Antibiotic Resistance Antibiotics Biochemical Biochemistry Biological Cells Complex Custom Data Defect Development Engineering Ensure Enzymes Family Fluorescence Health Human Hydrolysis Individual Interdisciplinary Study Investigation Leucine Life Link Maps Modeling Molecular Molecular Biology Nature Organism Outcome Pathway interactions Pharmacologic Substance Physiological Protein Biosynthesis Proteins RNA-Protein Interaction Reaction Research Resolution Ribosomes Roentgen Rays Site Social Welfare Specificity Structure Technology Therapeutic Transfer RNA Transfer RNA Aminoacylation Translations Work X-Ray Crystallography adenylate antimicrobial drug base computational chemistry deacylation design enzyme pathway in vivo Model innovation insight interdisciplinary approach leucine-tRNA molecular dynamics mutant nervous system disorder novel polypeptide research study single molecule single-molecule FRET tool

项目摘要

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases (aaRSs) comprise a family of up to twenty two 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, which guards against neurological disease. 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 amino 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 has historically complicated investigations to determine their respective molecular basis. New models for leucyl-tRNA synthetase (LeuRS) have been developed to isolate pre-transfer editing activities for detailed mechanistic investigations. In addition, pre-transfer editing can be dependent on tRNA. It is hypothesized that tRNA translocation between the synthetic and editing domains controls the enzyme's pathway choice between aminoacylation, post-transfer editing, and pre-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computation, single molecule flourescence, biochemical, and molecular biology approaches to investigate tRNA translocation and the molecular basis of the enzyme- tRNA transition that forms stable aminoacylation and editing complexes. It will also characterize pre-transfer editing and its physiological impact on the cell. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs, especially the LeuRS editing site, 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 tools in medicinal and technological applications.

描述（由申请人提供）：氨基酰基-TRNA合酶（AARSS）组成了一个多达22个酶的家族，这些酶对每个生物体都是必不可少的。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的准确底物识别，后者可以防止神经疾病。一些AARS已开发出编辑机制来纠正滥用的氨基酸。这些编辑AARS通过对两种底物激活的氨基酰基 - 亚戊酸酯（氨基酸的“前转移”到tRNA）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似乎是通过转移前后编辑的混合物来运作的，该编辑的混合物历史上已经进行了复杂的研究以确定其各自的分子基础。已经开发了亮氨基-TRNA合成酶（LEURS）的新模型，以分离转移预编辑活动以进行详细的机械研究。另外，转移的编辑可以取决于tRNA。假设合成域和编辑域之间的tRNA易位控制氨基化，转移后编辑和转移前编辑之间的酶选择。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，单分子粉，生化和分子生物学方法，以研究tRNA易位以及形成稳定的氨基氨基氨基化和编辑复合物的酶过渡的分子基础。它还将表征转移的预编辑及其对细胞的生理影响。对编辑机制的详细了解将使正在进行的药物研究受益于AARSS，尤其是Leurs编辑地点，作为抗生素开发的目标。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学或工具。