tRNA Synthesis Fidelity Mechanisms

描述(由申请人提供)：氨基酰-tRNA合成酶(AARs)由20个酶家族组成，这些酶对每个活着的有机体都是必不可少的。每种酶识别一种同源氨基酸，并将其共价连接到正确的tRNA上。“带电的”tRNA然后转移核糖体上的氨基酸，以特定的方式并入不断增长的多肽链。蛋白质合成的保真度完全依赖于AARSS对底物的准确识别。一些AARS已经开发了编辑机制来纠正未被激活的氨基酸。这些编辑的AARs通过两种底物中的任何一种的水解来清除错误的氨基酸--失活的氨基酰基腺苷酸盐(将氨基酸预转移到tRNA)或错酰化的AA-tRNA(转移后)。尽管这些机制中的一种可能占主导地位，但大多数编辑的AAR似乎是通过转移前和转移后编辑的混合来操作的，这使得确定它们各自的分子基础的研究变得复杂。大肠杆菌亮氨酰-tRNA合成酶(LEUR)是独一无二的，因为它只通过后转移机制进行编辑。在过去的资金周期中，转移后编辑活动被取消，在大肠杆菌中通过有限数量的突变激活了转移前编辑途径。因此，大肠杆菌野生型和突变型LEUR提供了一个强大的模型来分离两种AARS保真度机制，并表征转移前和/或转移后编辑特有的分子决定因素。这项建议概述了一个跨学科的研究计划，该计划结合了X射线结晶学、计算、生化和分子生物学的方法，以研究转移前编辑中错误激活的氨基酰腺苷中间体和转移后编辑中错误充电的tRNA的易位机制。它还将确定AARS对翻译保真度和细胞活力的生理影响。对编辑机制的详细了解将有助于正在进行的药物研究，这些研究将利用AARS作为抗生素开发的目标。它还将使AARs能够重新设计，以激活替代氨基酸，并将其整合到定制设计的蛋白质中。这些新型蛋白质可作为治疗药物或在医学和技术应用中的重要工具。

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases (aaRSs) comprise a family of twenty enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates ("pre-transfer" of arnino acid to tRNA) or misacylated aa- tRNA ("post-transfer"). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which complicates investigations to determine their respective molecular basis. E. coli leucyl-tRNA synthetase (LeuRS) is unique because it edits exclusively by a post-transfer mechanism. In the past funding cycle, the post-transfer editing activity was abolished and a pre-transfer editing pathway activated in E. coli LeuRS by a limited number of mutations. Thus, the E. coli wild-type and mutant LeuRS provide a powerful model to segregate the two aaRS fidelity mechanisms and characterize molecular determinants that are specific to pre- and/or post-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computational, biochemical, and molecular biology approaches to investigate translocation mechanisms for misactivated aminoacyl- adenylate intermediates in pre-transfer editing and mischarged tRNAs in post-transfer editing. It will also determine the physiological impact of the aaRSs on translational fidelity and cell viability. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.