tRNA Synthetase Fidelity Mechanisms

描述(申请人提供)：氨基酰-tRNA合成酶包括一个 由二十种酶组成的家族，这些酶对每个活着的有机体来说都是必不可少的。每个人 酶识别单一的同源氨基酸，并将其共价连接到 正确的tRNA。“带电的”trna随后转移核糖体上的氨基酸。 用于特定地结合到不断增长的多肽链中。的忠诚度 蛋白质的合成完全依赖于对底物的准确识别 TRNA合成酶。一些tRNA合成酶已经开发出编辑机制 纠正未被激活的氨基酸。这一校对步骤提高了保真度 幅度高达两个数量级。然而，人们对此知之甚少 识别未被激活的氨基酸或水解性切割机制。 该提案概述了一个跨学科的研究计划，该计划结合了 用计算、生化和分子生物学的方法来鉴定 编辑活性位点并阐明其分子机制 亮氨酰(Leu-)tRNA合成酶作为一种新的tRNA依赖的编辑活性 模特。亮氨酸酶的编辑活动由不同的物种特异性产生 机械装置。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进行编辑，而酵母 酶通过“预转移”机制发挥作用，该机制可使失活的酶 腺苷中间体。新出现的假说表明，前和 传输后编辑活动站点可能是不同的。初步数据为 标识传输后编辑活动站点的描述。后续 在特定目标的拟议工作我将绘制和描绘分子 大肠杆菌Leu-tRNA合成酶中这个编辑活性部位的决定因素。一个 还概述了一系列实验，以确定和表征 使用酵母Leu-tRNA合成酶预转编辑活性部位。具体而言 目的第二，提出了确定哪些非同源氨基酸的战略计划。 威胁Leu-tRNA合成酶的保真度，使该酶保持 正在编辑的活动站点。最后，互补性实验将检验 编辑缺陷trna合成酶对细胞活力的生理影响。 详细了解tRNA合成酶的编辑机制，特别是 那些特定于物种的，将有利于正在进行的药物研究 正在积极探索tRNA合成酶作为抗生素的靶标 发展。它还将使tRNA合成酶重新工程以激活 可替代氨基酸加入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药物的重要工具 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.