tRNA Synthesis Fidelity Mechanisms

描述（由申请人提供）：氨酰-tRNA合成酶（aaRS）包括一个由20种酶组成的家族，这些酶是每种生物体所必需的。每种酶识别一个单一的同源氨基酸，并将其共价连接到正确的tRNA上。然后，“带电”的tRNA将核糖体上的氨基酸转移到生长中的多肽链中。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错误激活的氨基酸。这些编辑aaRS通过水解两种底物中的任一种-错误活化的氨酰基-腺苷酸（氨基酸向tRNA的“转移前”）或错误酰化的aa-tRNA（“转移后”）来清除错误的氨基酸。虽然这些机制之一可能占主导地位，但大多数编辑的aaRS似乎是通过转移前和转移后编辑的混合物来操作的，这使得确定其各自分子基础的研究变得复杂。E.大肠杆菌亮氨酰-tRNA合成酶（LeuRS）是独特的，因为它仅通过转移后机制进行编辑。在过去的资助周期中，在E. coliLeuRS通过有限数量的突变。因此，E.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阿尔斯保真度机制，并表征特异于转移前和/或转移后编辑的分子决定簇。该提案概述了一个跨学科的研究计划，该计划结合了X射线晶体学，计算，生物化学和分子生物学方法，以研究转移前编辑中错误激活的氨酰腺苷酸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利于正在进行的利用aaRS作为抗生素开发靶点的药物研究。它还将使aaRS的重新工程化能够激活替代氨基酸以并入定制设计的蛋白质中。这些新的蛋白质可用作治疗剂或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.