TRANSFER RIBONUCLEIC ACID STRUCTURE AND FUNCTION

本研究的目的是确定序列和结构 tRNA被蛋白质识别所需的元件。 我们的特定 兴趣是对E.氨酰-tRNA法 合成酶和tRNA修饰酶，tRNA（尿嘧啶-5- ）甲基转移酶。 对这些相互作用的详细了解将 澄清有关蛋白质合成保真度的问题， tRNA修饰的机制，以及修饰的核苷在 tRNA功能 tRNAVal的元件对于其被 缬氨酰-tRNA合成酶（VRS）将通过检查充电来确定 体外转录合成tRNAVal变体的动力学 （克隆的）突变体tRNA基因。 合成酶的最低要求 识别将探索与tRNA片段含有必要的 tRNAVal的识别元件。 野生型和突变型的结构 tRNAVal将通过用标记的tRNA的19 F NMR光谱来探测， 通过掺入5-氟尿嘧啶（FUra）代替尿嘧啶来产生氟。 氟化的tRNAVal非常适合此类研究，因为它保留了 功能齐全，包含高灵敏度19 F探针， 在tRNA分子的所有螺旋茎和环中， 可以监测局部结构变化。 化合物19 F NMR谱 （FUra）tRNAVal显示14个掺入的氨基酸中的每一个的解析共振。 Fura残留。 既然已经分配了其中的每一个，我们计划使用 二维异源NOE（HOESY）技术检测反密码子 循环结构 19 F NMR光谱也将用于比较 （FUra）tRNAVal和突变体与同源物和非同源物相互作用 合成酶，以帮助澄清这些酶识别方式的差异 tRNA。 tRNAVal与tRNA（尿嘧啶-5-）甲基转移酶的相互作用， 还将随后进行19 F NMR，以探索诱导的构象变化 在tRNA的酶，并阐明酶的细节， 共价tRNA-蛋白质反应的结构和机制 中间体的 这些调查的结果将提供基本的 关于蛋白质-tRNA相互作用和tRNA之间关系的信息 结构和功能。 关于纳入FURA的后果的研究 这将有助于更好地了解抗肿瘤 氟嘧啶的作用及其细胞毒性。

The goals of this research are to define the sequence and structural elements of tRNA required for its recognition by proteins. Our particular interest is the recognition of E. coli tRNAVal 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 tRNAVal essential for its recognition by valyl-tRNA synthetase (VRS) will be determined by examining the charging kinetics of tRNAVal variants synthesized in vitro by transcription from (cloned) mutant tRNA genes. Minimal requirements for synthetase recognition will be explored with tRNA fragments containing the essential recognition elements of tRNAVal. The structure of wild-type and mutant tRNAVal will be probed by 19F NMR spectroscopy of tRNAs labeled with fluorine by incorporation of 5-fluorouracil (FUra) in place of uracil. Fluorinated tRNAVal is ideally suited for such studies because it remains fully functional and contains highly sensitive 19F probes distributed throughout all the helical stems and loops of the tRNA molecule where they can serve to monitor local structural changes. The 19F NMR spectrum of (FUra)tRNAVal shows a resolved resonance for each of the 14 incorporated FUra residues. Now that each of these has been assigned, we plan to use two-dimensional heteronuclear NOE (HOESY) techniques to examine anticodon loop structure. 19F NMR spectroscopy will also be used to compare the interaction of (FUra)tRNAVal and mutants with cognate and non-cognate synthetases, to help clarify differences in the way these enzymes recognize tRNA. The interaction of tRNAVal with tRNA (uracil-5-)methyltransferase, will also be followed by 19F NMR, to explore conformational changes induced in the tRNA by the enzyme, and to elucidate details of the enzyme mechanisms and of the structures of covalent tRNA-protein reaction intermediates. The results of these investigations will provide basic information on protein-tRNA interactions and on the relation between tRNA structure and function. Study of the consequences of FUra incorporation into tRNA should contribute to a better understanding of the antitumor effects of fluoropyrimidines and their cytotoxicity.