Structure and Mechanism of Class II tRNA Synthetases

描述(由申请人提供)：氨基酰-tRNA合成酶(AARs)是解码所有活细胞中遗传信息的关键酶。尽管它们的发现相对较早，最近也进行了广泛的结构表征，但它们如何区分密切相关的氨基酸和转移RNA底物仍在积极研究中。AARS的基本问题包括：1)同一类别的酶之间是否有共同的一般机制特征；2)氨基酰化反应速率受限的确切步骤(S)，以及氨基酸专一性是否在该步骤中起调节作用；3)转移RNA中的特定识别元件如何发挥作用；以及4)防止未被激活和错误酰化的氨基酸被引入细胞蛋白质的具体机制。为了解决这些问题，我们将利用在前一个资金时期开发的快速猝灭和停止流动荧光方法来测量氨基酸激活和氨基酰化反应中的基本步骤的速率，从而检验存在II类Aars超家族共同的机制特征的假设。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调的氨酰化机制的共性；2)通过定义tRNA同一性决定因素对氨酰化作用最深刻的基本步骤，阐明tRNA识别的分子基础；3)利用固有的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合成酶的结构变化联系起来；以及4)通过测量基本步骤的速率和编辑类的结合热力学来确定苏氨酰-tRNA合成酶的编辑机制。对氨基酰-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制的进化和操作中的基础作用中得出了它们的相关性。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群组成，GCN2亚家族是一种新的与脑功能相关的调节蛋白。

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.