Specificity in the Synthesis of Aminoacyl-tRNA

描述(由申请人提供)：将在各种实验系统中研究氨基酰-tRNA合成的特异性的潜在机制。首先，最近确定的大肠杆菌半胱氨酰-tRNA合成酶的结构将被用作探索该系统中氨基酸和tRNA选择性的独特机制的基础。将研究锌介导的构象开关控制tRNA3‘端在活性部位的位置，并将在人和大肠杆菌CysRS的互补研究中探索形状选择性tRNA识别的起源。接下来，我们将研究大肠杆菌谷氨酰tRNA合成酶中氨基酸和tRNA特异性偶联的机理基础。在这种情况下，主要的方法将是应用新开发的瞬变动力学方法。这些实验的结果将为通过合理突变设计氨基酸特异性开关提供信息，以期引入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依赖的氨基转移酶将错酰化的tRNA转化为适合蛋白质合成的底物的机制。这里的实验将集中在建立tRNA识别元件的身份和反应途径的某些方面。阐明诱导FIT和间接读出机制如何控制tRNA和模型tRNA合成酶中的氨基酸特异性，将有助于理解更复杂的粒子(如核糖体)中的此类过程。此外，基于对人类合成酶和细菌合成酶的区分，以及细菌tRNA氨基转移酶的性质，开发新的抗菌化合物具有巨大的潜力，而细菌tRNA氨基转移酶没有人类的对应物。最后，设计tRNA合成酶来扩展遗传密码可能会打开基于蛋白质的新疗法的大门，并有可能影响许多人类疾病的疗法的发展。

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.