tRNA Synthetase Fidelity Mechanisms

描述（由申请人提供）： 氨酰-tRNA合成酶（aaRSs）包括一个多达22种酶的家族，它们对每个生物体都是必需的。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，这可以预防神经疾病。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何一种-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，这在历史上使研究复杂化以确定其各自的分子基础。已经开发了亮氨酰-tRNA合成酶（LeuRS）的新模型，以分离转移前的编辑活动，用于详细的机制研究。此外，转移前编辑可以依赖于tRNA。假设合成和编辑结构域之间的tRNA易位控制酶在氨酰化、转移后编辑和转移前编辑之间的途径选择。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，单分子荧光，生物化学和分子生物学方法，以研究tRNA易位和形成稳定的氨酰化和编辑复合物的酶- tRNA转变的分子基础。它还将表征转移前编辑及其对细胞的生理影响。对编辑机制的详细了解将有利于正在进行的利用aaRS，特别是LeuRS编辑位点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药物和技术应用中的治疗剂或工具。

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases (aaRSs) comprise a family of up to twenty two 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, which guards against neurological disease. 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 amino 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 has historically complicated investigations to determine their respective molecular basis. New models for leucyl-tRNA synthetase (LeuRS) have been developed to isolate pre-transfer editing activities for detailed mechanistic investigations. In addition, pre-transfer editing can be dependent on tRNA. It is hypothesized that tRNA translocation between the synthetic and editing domains controls the enzyme's pathway choice between aminoacylation, post-transfer editing, and pre-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computation, single molecule flourescence, biochemical, and molecular biology approaches to investigate tRNA translocation and the molecular basis of the enzyme- tRNA transition that forms stable aminoacylation and editing complexes. It will also characterize pre-transfer editing and its physiological impact on the cell. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs, especially the LeuRS editing site, 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 tools in medicinal and technological applications.