Structure and Mechanism of Class II tRNA Synthetases

描述(申请人提供)：氨基酰-tRNA合成酶是必需的 在对所有生物系统中的遗传信息进行解码时。尽管他们 相对较早的发现和最近广泛的结构表征， 关于它们的机制和分子细节的重要问题 功能仍有待解决。在先前资助的周期内， 首席研究员对腺化反应有了相当完整的了解。 组二酰-tRNA合成酶催化反应的结晶学和核磁共振 分子遗传学，确定酶的可移动区域对 催化作用。他进一步证明，tRNA在多个步骤中与酶结合， 基序2环内的残基对鉴别子碱基是重要的 专一性。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，具有一个 在组氨酸生物合成调控中的作用。他还提供了第一个 在ThrRS中进行编辑的证据。与tRNA相关的一些重要问题 合成酶的功能尚待解决，其中包括结构蛋白的作用 反应过程中酶和tRNA的变化。在这个项目中 更新，首席研究员将研究tRNA的动力学 合成酶：组氨酸和苏氨酸系统中的tRNA相互作用 荧光分析监测腺化的各个步骤和 氨酰化反应。具体目标是：1)确定酶的作用 两类氨基酰tRNA腺基化反应的构象动力学 合成酶的瞬时动力学分析；2)测定合成酶的产率 对氨基酰化反应进行了瞬时动力学分析，考察了 在tRNA结合过程中有重要的中间体；3)调查 用瞬时动力学分析法研究ThrRS中tRNA编辑的基础 建立氨基酸选择的定量依据和氨基酸选择的比率 野生型和突变酶中的苏氨酸化和丝氨酸化。穿过 氨基酰化反应中荧光变化与机械性步骤的关系 在HisRS和ThrRS的催化下，首席研究员希望获得 洞察识别核的酶如何识别的基本问题 序列特定方式的酸利用结合诱导的构象 改变以促进催化。

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.