Specificity in the Synthesis of Aminoacyl-tRNA

氨酰基-tRNA 合成的特异性

• 批准号: 7073997
• 负责人: JOHN J. PERONA
• 金额: $ 26.59万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-07 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073997
• 关键词: X ray crystallography; active sites; aminoacid tRNA ligase; bacterial proteins; biochemistry; chemical binding; chemical kinetics; conformation; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; molecular shape; protein engineering; protein structure function; site directed mutagenesis; structural biology; transfer RNA

DESCRIPTION (provided by applicant): Mechanisms underlying the specificity of aminoacyl-tRNA synthesis will be studied in a variety of experimental systems. First, recently determined structures of E. coli cysteinyl-tRNA synthetase will be used as a basis for exploring unique mechanisms underlying the selectivities for amino acid and tRNA in that system. A zinc-mediated conformational switch controlling placement of the tRNA 3'-end in the active site will be studied, and the origins of shape-selective tRNA recognition will be explored in complementary studies of human and E. coli CysRS. Next, the mechanistic basis for the coupling of amino acid and tRNA specificities will be studied in E. coli glutaminyl-tRNA synthetase. In this case the primary approach will be the application of newly-developed transient kinetic methods. Results of these experiments will inform the design of amino acid specificity switches by rational mutagenesis, with a view towards introduction of activity towards noncognate and nonstandard amino acids. Finally, the mechanisms by which the H. pylori tRNA- dependent amidotransferase converts misacylated tRNAs into suitable substrates for protein synthesis will be studied. Experiments here will focus on establishing the identities of the tRNA recognition elements and some aspects of the reaction pathway. The elucidation of how induced fit and indirect readout mechanisms control tRNA and amino acid specificities in model tRNA synthetases will be relevant to understanding such processes in more complex particles such as the ribosome. Further, there is great potential for developing novel antimicrobial compounds based both on discrimination between human and bacterial synthetases, and on the properties of bacterial tRNA amidotransferases, which possess no human counterparts. Finally, engineering of tRNA synthetases to expand the genetic code may open the door to novel protein-based therapeutics and has the potential to impact the development of therapies for many human diseases.

描述（由申请人提供）：将在各种实验系统中研究氨酰-tRNA合成特异性的机制。首先，最近确定的大肠杆菌半胱氨酰-tRNA 合成酶的结构将用作探索该系统中氨基酸和 tRNA 选择性的独特机制的基础。将研究控制 tRNA 3' 端在活性位点中的位置的锌介导的构象开关，并将在人类和大肠杆菌 CysRS 的互补研究中探索形状选择性 tRNA 识别的起源。接下来，将研究大肠杆菌谷氨酰胺酰-tRNA 合成酶中氨基酸和 tRNA 特异性偶联的机制基础。在这种情况下，主要方法将是应用新开发的瞬态动力学方法。这些实验的结果将为通过合理诱变设计氨基酸特异性开关提供信息，以期引入针对非同源和非标准氨基酸的活性。最后，将研究幽门螺杆菌 tRNA 依赖性酰胺转移酶将异酰基化 tRNA 转化为适合蛋白质合成的底物的机制。这里的实验将集中于确定 tRNA 识别元件的身份和反应途径的某些方面。阐明诱导拟合和间接读出机制如何控制模型 tRNA 合成酶中的 tRNA 和氨基酸特异性，将有助于理解更复杂的颗粒（如核糖体）中的此类过程。此外，基于人类和细菌合成酶之间的区别以及细菌 tRNA 酰胺转移酶的特性，开发新型抗菌化合物具有巨大的潜力，而细菌 tRNA 酰胺转移酶不具有人类对应物。最后，通过工程化 tRNA 合成酶来扩展遗传密码可能会为新型蛋白质疗法打开大门，并有可能影响许多人类疾病疗法的开发。