Structure and Mechanism of Class II tRNA Synthetases

II类tRNA合成酶的结构和机制

基本信息

批准号：
7892234
负责人：
CHRISTOPHER S FRANCKLYN
金额：
$ 19.94万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7892234
关键词：
Ablation Active Sites Address Affinity Alanine-tRNA Ligase Amino Acids Amino Acids Activation Amino Acyl Transfer RNA Amino Acyl-tRNA Synthetases Aminoacylation Antibiotics Anticodon Binding Biological Assay Brain Calorimetry Catalysis Cells Chemistry DNA-Directed DNA Polymerase Development Discrimination Elements Enzymes Evolution Face Family Fluorescence Fluorescence Resonance Energy Transfer Funding Gene Expression Genetic Histidine Histidine-tRNA Ligase Indium Investigation Kinetics Knowledge Length Life Measurement Measures Mediating Minor Groove Mitochondria Modeling Molecular Movement Mutagenesis Operative Surgical Procedures Organism Pathway interactions Process Protein Biosynthesis Proteins RNA, Transfer, Amino Acid-Specific Reaction Reading Research Personnel Ribosomes Role Serine Specificity Structure Subgroup System Testing Thermodynamics Threonine-Specific tRNA Threonine-tRNA Ligase Titrations Transfer RNA Tryptophan Work analog base deacylation genetic regulatory protein in vivo inorganic phosphate mutant novel prevent research study stem stopped-flow fluorescence

项目摘要

DESCRIPTION (provided by applicant): Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in the decoding of genetic information in all living cells. Despite their relatively early discovery and recent extensive structural characterization, how they achieve discrimination between closely related amino acid and transfer RNA substrates is under active investigation. Among the fundamental questions for the aaRSs are i) whether there are general mechanistic features shared among enzymes in the same class; ii) the precise step(s) at which aminoacylation is rate limited, and whether amino acid specificity is mediated at that step; iii) how specific recognition elements in transfer RNAs exert their effects; and iv) the specific mechanisms that prevent misactivated and misacylated amino acids from being introduced into cellular proteins. To address these questions, we will make use of rapid quench and stopped flow fluorescence approaches developed during the previous funding period to measure rates of elementary steps in the amino acid activation and aminoacylation reactions, and thereby test the hypothesis that mechanistic features common to the class II aaRS superfamily exist. Our aims include: 1) determining the generality of a substrate-assisted and concerted aminoacylation mechanism discovered in histidyl-tRNA synthetases by investigations of threonyl- and alanyl-tRNA synthetases; 2) clarifying the molecular basis of tRNA recognition by defining the elementary steps at which tRNA identity determinants exert their most profound effects on aminoacylation; 3) correlating elementary steps in the aminoacylation pathway with structural changes in threonyl- and histidyl-tRNA synthetase, making use of intrinsic fluorescence and resonance energy transfer; and 4) determining the mechanism of editing in threonyl- tRNA synthetases by measurement of the rates of elementary steps and the binding thermodynamics of editing analogs. Investigations of aminoacyl-tRNA synthetases draw their relevance from the universal presence of these enzymes in all living systems, and their fundamental role in the evolution and operation of the translational machinery. Differences between prokaryotic and eukaryotic enzymes have been exploited in the development of new antibiotics, as well as the incorporation of unnatural amino acids into proteins. The histidyl-tRNA synthetase family is composed of three subgroups with regulatory functions, and the GCN2 subfamily is emerging as a novel regulatory protein with a role in brain function.

描述（由申请人提供）：氨基酰基-TRNA合成酶（AARSS）是所有活细胞中遗传信息解码的必需酶。尽管它们相对较早地发现和最近的广泛结构表征，但如何实现密切相关的氨基酸和转移RNA底物的区分仍在积极研究中。在AARS的基本问题中，i）同一类酶之间是否共享一般的机械特征； ii）氨基化速率限制的确切步骤，以及在此步骤中是否介导氨基酸特异性； iii）转移RNA中的特定识别要素如何发挥其作用； iv）防止被误导和放错氨基酸引入细胞蛋白的特定机制。为了解决这些问题，我们将利用在上一个资金期间开发的快速淬火和停止流动荧光方法，以衡量氨基酸激活和氨基酰化反应中基本步骤的速率，从而检验了II AARS II AARS II ARS超级家庭的机械特征的假设。我们的目的包括：1）确定在组酰基-TRNA合成酶中发现的底物辅助和协同的氨基酰化机制的一般性，该机制是通过研究三酰基和丙酰基-TRNA合成酶的； 2）通过定义tRNA身份决定因素对氨基酰化作用最深远的基本步骤来阐明tRNA识别的分子基础； 3）将氨基酰化途径中的基本步骤与三酰基和组酰基-TRNA合成酶的结构变化相关联，利用了内在的荧光和共振能传递； 4）通过测量基本步骤的速率和编辑类似物的结合热力学来确定在三季度合成酶中编辑的机制。对氨基酰基-TRNA合酶的研究从这些酶在所有生物系统中的普遍存在中及其在转化机械的演化和运行中的基本作用中汲取了相关性。在新抗生素的发展以及将非天然氨基酸掺入蛋白质中，原核和真核酶之间的差异已被利用。组酰基-TRNA合成酶家族由具有调节功能的三个亚组组成，而GCN2亚家族正在成为一种新型调节蛋白，在脑功能中起作用。