Structure and Mechanism of Class II tRNA Synthetases

II类tRNA合成酶的结构和机制

• 批准号: 7610970
• 负责人: CHRISTOPHER S FRANCKLYN
• 金额: $ 31.36万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2010-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7610970
• 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

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）：氨酰基-tRNA 合成酶（aaRS）是所有活细胞中遗传信息解码的必需酶。尽管它们的发现相对较早并且最近进行了广泛的结构表征，但它们如何实现密切相关的氨基酸和转移 RNA 底物之间的区分仍在积极研究中。 aaRS 的基本问题包括 i) 同一类酶之间是否存在共同的一般机制特征； ii) 氨酰化受到速率限制的精确步骤，以及该步骤是否介导氨基酸特异性； iii) 转移RNA中的特定识别元件如何发挥作用； iv) 防止错误激活和错误酰化氨基酸被引入细胞蛋白质的具体机制。为了解决这些问题，我们将利用上一个资助期间开发的快速猝灭和停流荧光方法来测量氨基酸激活和氨酰化反应中基本步骤的速率，从而检验 II 类 aaRS 超家族存在共同机制特征的假设。我们的目标包括：1) 通过研究苏氨酰和丙氨酰 tRNA 合成酶，确定组氨酰 tRNA 合成酶中发现的底物辅助和协同氨酰化机制的普遍性； 2) 通过定义 tRNA 身份决定因素对氨酰化发挥最深远影响的基本步骤，阐明 tRNA 识别的分子基础； 3)利用内在荧光和共振能量转移，将氨酰化途径中的基本步骤与苏氨酰-和组氨酰-tRNA合成酶的结构变化相关联； 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苏氨酰-tRNA合成酶中的编辑机制。对氨酰基-tRNA 合成酶的研究源于这些酶在所有生命系统中的普遍存在，以及它们在翻译机制的进化和操作中的基本作用。原核酶和真核酶之间的差异已被用于开发新抗生素以及将非天然氨基酸掺入蛋白质中。组氨酰-tRNA合成酶家族由具有调节功能的三个亚群组成，其中GCN2亚家族正在成为一种在脑功能中发挥作用的新型调节蛋白。