Structural Basis of Transfer Ribonucleic Acid Function

本研究的目的是确定转移核糖核酸（tRNA）被蛋白质识别所需的序列和结构元素。特别令人感兴趣的是大肠杆菌tRNA对氨基酸缬氨酸（tRNAVa1）的识别是通过氨基酰基tRNA合成酶和tRNA修饰酶tRNA（尿嘧啶-5-）甲基转移酶实现的。对这些相互作用的详细了解将澄清有关蛋白质合成的保真度、tRNA修饰的机制以及修饰核苷在tRNA功能中的作用等问题。tRNAVa1被缬氨酸- trna合成酶（VRS）识别所必需的tRNAVa1元素将通过检查tRNAVa1变体的反应动力学来确定。对合成酶识别的最低要求将与含有tRNAVa1基本识别元件的tRNA片段一起探讨。这些突变体trna的结构将通过氟化tRNAVa1标签的19F核磁共振光谱进行探测。氟化tRNAVa1非常适合此类研究，因为它保持完整的功能，并且包含分布在tRNA分子的所有螺旋茎和环中的高度敏感的19F探针，在那里它们可以监测局部结构变化。

The goals of this research are to define the sequence and structural elements of transfer ribonucleic acid (tRNA) required for its recognition by proteins. Of particular interest is the recognition of E. coli tRNA for amino acid valine (tRNAVa1) by aminoacyl-tRNA synthetases and by the tRNA modifying enzyme, tRNA (uracil-5-)methyltransferase. Detailed understanding of these interactions will clarify questions concerning the fidelity of protein synthesis, the mechanisms of tRNA modification, and the role of modified nucleosides in tRNA function. The elements of tRNAVa1 essential for its recognition by valyl-tRNA synthetase (VRS) will be determined by examining the kinetics of reaction of tRNAVa1 variants. Minimal requirements for synthetase recognition will be explored with tRNA fragments containing the essential recog- nition elements of tRNAVa1. The structure of these mutant tRNAs will be probed by 19F NMR spectroscopy of tRNAVa1 labe- led with fluorine. Fluorinated tRNAVa1 is ideally suited for such studies because it remains fully functional and contains highly sensitive 19F probes distributed throughout all the helical stems and the loops of the tRNA molecule where they can serve to monitor local structural changes.