Structure and Mechanism of Class II tRNA Synthetases

II类tRNA合成酶的结构和机制

• 批准号: 6621603
• 负责人: CHRISTOPHER S FRANCKLYN
• 金额: $ 28.57万
• 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 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621603
• 关键词: X ray crystallography; active sites; acylation; aminoacid tRNA ligase; conformation; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence resonance energy transfer; fluorescent dye /probe; histidine; intermolecular interaction; protein binding; protein purification; site directed mutagenesis; threonine; transfer RNA

DESCRIPTION (provided by applicant): Aminoacyl- tRNA synthetases are essential in the decoding of genetic information in all living systems. Despite their relatively early discovery and recent extensive structural characterization, significant questions concerning the details of their mechanisms and molecular functions remain to be addressed. During the previously funded cycle, the principal investigator developed a fairly complete picture of the adenylation reaction catalyzed by histididyl-tRNA synthetase using crystallography and molecular genetics, identifying mobile regions of the enzyme important for catalysis. He further showed that tRNA binds to the enzyme in multiple steps, and that residues within the motif 2 loop are important for discriminator base specificity. Other studies investigated evolutionary aspects of the HisRS family, including the discovery of a new family of HisRS-like proteins with a role in the regulation of histidine biosynthesis. He also provided the first evidence for editing in ThrRS. A number of important questions related to tRNA synthetase function remain to be resolved, including the role of structural changes in both the enzyme and the tRNA during the reaction. In this project renewal, the principal investigator will investigate the dynamics of tRNA synthetase:tRNA interactions in the histidine and threonine systems by use of fluorescence assays to monitor individual steps in the adenylation and aminoacylation reactions. Specific aims are to: 1) define the role of enzyme conformational dynamics in the adenylation reaction in two class aminoacyl-tRNA synthetases by transient kinetic analysis; 2) determine the rate of the aminoacylation reaction by transient kinetic analysis and investigate whether there are significant intermediates in the tRNA binding process; 3) investigate the basis of tRNA editing in ThrRS by using transient kinetic analysis to establish the quantitative basis of amino acid selection and the rates of threonylation and serylation in wild type and mutant enzymes. Through correlation of fluorescence changes to mechanistic steps in the aminoacylation reaction catalyzed by HisRS and ThrRS, the principal investigator hopes to gain insight into the fundamental question of how enzymes that recognize nucleic acids in a sequence specific fashion utilize binding-induced conformational changes to promote catalysis.

描述（由申请人提供）：氨酰-tRNA合成酶是必需的 在所有生命系统中的遗传信息解码中。尽管他们 相对较早的发现和最近广泛的结构表征， 关于其机制和分子的细节的重要问题 职能仍有待解决。在上一个供资周期， 首席研究员开发了一个相当完整的图片的腺苷酸化 使用晶体学由组氨酰-tRNA合成酶催化的反应， 分子遗传学，确定酶的移动的区域， 催化作用他进一步表明，tRNA与酶的结合是多步骤的， 并且基序2环内的残基对于C37BL是重要的 的特异性其他研究调查了HisRS的进化方面 家族，包括发现一个新的HisRS样蛋白家族， 在组氨酸生物合成调节中的作用。他还提供了第一个 在ThrRS中编辑的证据。与tRNA相关的一些重要问题 合成酶的功能仍有待解决，包括结构的作用， 酶和tRNA在反应过程中的变化。在这个项目中 更新，主要研究者将研究tRNA的动态 组氨酸和苏氨酸系统中的合成酶：tRNA相互作用 荧光测定以监测腺苷酸化中的各个步骤， 氨酰化反应。具体目标是：1）确定酶的作用 两类氨酰-tRNA腺苷酸化反应的构象动力学 合成酶的瞬时动力学分析; 2）确定的速率， 氨酰化反应的瞬态动力学分析，并研究是否 在tRNA结合过程中存在重要的中间体; 3）研究 通过使用瞬时动力学分析， 建立了氨基酸选择的定量依据， 野生型和突变酶中的苏氨酰化和丝氨酰化。通过 荧光变化与氨酰化中的机械步骤的相关性 由HisRS和ThrRS催化的反应，主要研究者希望获得 深入了解酶如何识别核酸 酸以序列特异性方式利用结合诱导的构象 促进催化作用的变化。