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基- trna合成特异性的机制。首先，最近确定的大肠杆菌半胱氨酸-tRNA合成酶的结构将作为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tRNA 3'端在活性位点的位置，并在人类和大肠杆菌cyrs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合成酶中氨基酸与tRNA特异性耦合的机制基础。在这种情况下，主要的方法将是应用新开发的瞬态动力学方法。这些实验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转化为合适的底物用于蛋白质合成的机制。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颗粒（如核糖体）中的这一过程。此外，基于人类和细菌合成酶的区别，以及细菌tRNA氨基转移酶的特性，开发新的抗菌化合物具有很大的潜力，而细菌tRNA氨基转移酶在人类中没有对应物。最后，tRNA合成酶的工程扩展遗传密码可能会打开新的基于蛋白质的治疗方法的大门，并有可能影响许多人类疾病的治疗方法的发展。

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.