Structure and Mechanism of Class II tRNA Synthetases

描述（由申请人提供）：氨酰-tRNA合成酶（aaRS）是所有活细胞中解码遗传信息的必需酶。尽管他们相对较早的发现和最近广泛的结构表征，他们如何实现密切相关的氨基酸和转移RNA底物之间的歧视正在积极调查。aaRS的基本问题包括：i）是否存在同类酶之间共有的一般机制特征; ii）氨酰化速率受限的精确步骤，以及在该步骤是否介导氨基酸特异性; iii）转移RNA中的特异性识别元件如何发挥其作用;和iv）防止错误活化和错误酰化的氨基酸被引入细胞蛋白质的特定机制。为了解决这些问题，我们将利用快速淬灭和停流荧光的方法在以前的资助期间开发的氨基酸活化和氨酰化反应的基本步骤的测量率，从而测试的假设，即共同的II类阿尔斯超家族存在的机械功能。我们的目标包括：1）通过对苏氨酰-和丙氨酰-tRNA合成酶的研究，确定在组氨酰-tRNA合成酶中发现的底物辅助和协同氨酰化机制的一般性; 2）通过定义tRNA身份决定子对氨酰化发挥最深刻影响的基本步骤，阐明tRNA识别的分子基础; 3）利用固有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合成酶中的结构变化相关联; 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。氨酰-tRNA合成酶的研究从这些酶在所有生命系统中的普遍存在以及它们在翻译机器的进化和操作中的基本作用中得出它们的相关性。原核生物和真核生物酶之间的差异已被用于开发新的抗生素，以及将非天然氨基酸掺入蛋白质中。组氨酰-tRNA合成酶家族由三个具有调节功能的亚组组成，GCN 2亚家族正在成为一种新型调节蛋白，在大脑功能中发挥作用。

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.