tRNA Synthetase Fidelity Mechanisms

说明（由申请人提供）：氨酰-tRNA合成酶包含 由20种酶组成的家族，对每一种生物体都是必不可少的。每个 酶识别单个同源氨基酸，并将其共价连接到 正确的tRNA然后，“带电荷”的tRNA将氨基酸转移到核糖体上， 用于特异性掺入生长的多肽链中。的保真度 蛋白质合成完全依赖于精确的底物识别， tRNA合成酶。一些tRNA合成酶已经发展出编辑机制 来纠正错误激活的氨基酸这个校对步骤增加了保真度 相差两个数量级然而，人们对此知之甚少。 识别错误活化的氨基酸或水解裂解机制。 该提案概述了一个跨学科的研究计划， 计算，生物化学和分子生物学方法来识别 编辑活性位点，并阐明其分子机制。 使用亮氨酰（Leu-）tRNA合成酶作为新的tRNA依赖性编辑活性 模型亮氨酸酶的编辑活动是通过不同的物种特异性 机制等急诊大肠杆菌酶表现出明显的“转移后”活性， 需要将氨基酸转移到tRNA上进行编辑，而酵母 酶通过“预转移”机制发挥作用， 腺苷酸中间体。新出现的假说表明， 转移后编辑活性位点可以不同。初步数据 所描述的标识转移后编辑活动站点的标识符。后续 在具体目标中提出的工作我将绘制和描绘分子 E. coliLeu-tRNA合成酶。一 还概述了一系列实验，以确定和表征 使用酵母Leu-tRNA合成酶的预转移编辑活性位点。在特定 目的二，提出了一个战略计划，以确定哪些非同源氨基酸 威胁Leu-tRNA合成酶的保真度，使得该酶保持 一个活跃的编辑网站。最后，互补实验将测试 编辑缺陷型tRNA合成酶对细胞活力的生理作用。 详细了解tRNA合成酶编辑机制，特别是 那些物种特异性的，将有利于正在进行的药物研究， 积极探索tRNA合成酶作为抗生素的靶点 发展它还将使tRNA合成酶的重新工程能够激活 用于掺入定制设计的蛋白质的替代氨基酸。这些 新的蛋白质可以用作治疗剂或医学上的重要工具， 和技术应用。

DESCRIPTION (provided by applicant): The aminoacyl-tRNA synthetases 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 tRNA synthetases. Some tRNA synthetases have developed editing mechanisms to correct misactivated amino acids. This proofreading step increases fidelity by as much as two orders of magnitude. However, little is known about the recognition of misactivated amino acids or the hydrolytic cleavage mechanism. This proposal outlines an interdisciplinary research plan combining computational, biochemical, and molecular biology approaches to identify the editing active sites and elucidate the molecular mechanism of the tRNA-dcpendent editing activity using leucyl (Leu-) tRNA synthetase as a novel model. The leucine enzyme editing activities occur by distinct species-specific mechanisms. The E. coli enzyme exhibits a clear "post-transfer" activity that requires transfer of the amino acid to the tRNA for editing, while the yeast enzyme functions by a "pre-transfer" mechanism that hydrolyzes misactivated adenylate intermediates. Emerging hypotheses suggest that the pre- and post-transfer editing active sites may be distinct. Preliminary data are described that identifies the post-transfer editing active site. Subsequent proposed work in Specific Aim I will map and delineate the molecular determinants of this editing active site in E. coli Leu-tRNA synthetase. A series of experiments are also outlined to identify and characterize the pre-transfer editing active site using yeast Leu-tRNA synthetase. In Specific Aim II, a strategic plan is presented to determine which noncognate amino acids threaten the fidelity of Leu-tRNA synthetases such that this enzyme maintains an editing active site. Finally, complementation experiments will test the physiological effects of editing-defective tRNA synthetases on cell viability. A detailed understanding of the tRNA synthetase editing mechanism, particularly those that are species-specific, will benefit ongoing pharmaceutical research that is actively exploring tRNA synthetases as a target for antibiotic development. It will also enable reengineering of tRNA synthetases to activate alternative amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.