tRNA Synthetase Fidelity Mechanisms

tRNA 合成酶保真机制

• 批准号: 6526202
• 负责人: SUSAN A MARTINIS
• 金额: $ 22.55万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526202
• 关键词: Escherichia coli; active sites; aminoacid tRNA ligase; bacterial genetics; enzyme mechanism; fungal genetics; posttranscriptional RNA processing; site directed mutagenesis; transfer RNA; yeasts

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.

描述（由申请人提供）：氨基酰基-TRNA合成酶包括 对每个生物体至关重要的二十种酶家族。每个 酶识别单个同源氨基酸，并共价连接到 正确的tRNA。然后，“带电”的tRNA在核糖体处转移氨基酸 特定掺入不断增长的多肽链中。忠诚 蛋白质合成完全取决于准确的底物识别 tRNA合成酶。一些tRNA合成酶已经开发了编辑机制 纠正滥用的氨基酸。此校对步骤增加了保真度 多达两个数量级。但是，关于 识别滥用的氨基酸或水解裂解机制。 该建议概述了跨学科研究计划的结合 计算，生化和分子生物学方法来识别 编辑主动位置并阐明 tRNA-DCEDENTENT编辑活性使用leu--）tRNA合成酶作为一种新颖 模型。亮氨酸酶编辑活动通过不同的物种特异性发生 机制。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中进行编辑，而酵母 酶通过水解被滥用的“预先转移”机制起作用 腺苷酸中间体。新兴的假设表明，前和 转移后编辑活动站点可能是不同的。初步数据是 描述的是识别转移后编辑活动站点。随后的 提议的特定目的工作我将映射并描绘分子 大肠杆菌Leu-tRNA合成酶中此编辑活性位点的决定因素。一个 还概述了一系列实验以识别和表征 使用酵母Leu-tRNA合成酶进行预转移编辑活性位点。具体 AIM II，提出了一项战略计划，以确定哪些非同点氨基酸 威胁Leu-tRNA合成酶的保真度，以使该酶保持 编辑活动站点。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影响。 对TRNA合成酶编辑机制的详细理解，特别是 特定物种的人将受益于正在进行的药物研究 那是积极探索tRNA合成酶作为抗生素的靶标 发展。它还将使TRNA合成酶的重新设计能够激活 用于掺入定制设计的蛋白质中的替代氨基酸。这些 新型蛋白质可以用作药物治疗或重要工具 和技术应用。