tRNA Synthesis Fidelity Mechanisms

描述（由申请人提供）：氨基酰基trna合成酶（aaRSs）包括一个由20种酶组成的家族，这些酶对每个生物体都是必不可少的。每种酶识别一个同源氨基酸，并将其共价连接到正确的tRNA上。“带电”的tRNA随后将核糖体上的氨基酸转移到正在生长的多肽链中，以进行特定的结合。蛋白质合成的保真度完全依赖于aars对底物的准确识别。一些aars已经开发出编辑机制来纠正失活的氨基酸。这些编辑aars通过水解两种底物中的任何一种来清除错误的氨基酸——失活的氨基酰腺苷酸（“前转移”的arino酸到tRNA）或错酰基化的aa- tRNA（“后转移”）。尽管其中一种机制可能占主导地位，但大多数进行编辑的aars似乎是通过转移前和转移后编辑的混合操作的，这使得确定它们各自的分子基础的研究变得复杂。大肠杆菌亮氨酸- trna合成酶（LeuRS）是独特的，因为它完全通过转移后机制进行编辑。在过去的资助周期中，大肠杆菌LeuRS的转移后编辑活性被取消，转移前编辑途径被有限数量的突变激活。因此，大肠杆菌野生型和突变型LeuRS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/或转移后编辑特异性的分子决定因素。该提案概述了一个跨学科的研究计划，结合x射线晶体学、计算、生化和分子生物学方法，研究转移前编辑中失活的氨基酰腺苷酸中间体和转移后编辑中错载的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利于正在进行的药物研究，利用aars作为抗生素开发的靶点。它还将使aars重新工程，以激活替代氨基酸并入定制设计的蛋白质。这些新型蛋白可作为治疗药物或重要的药物和技术工具。

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.