Biophysical Studies on the Additive Effects of Modification in tRNA and rRNA

Nikonowicz，Edward P.MCB 0078501该项目的目标是在分子水平上了解tRNA转录后修饰过程的关键方面，包括修饰酶对分子特异性RNA识别的基础和RNA分子内修饰的功能基础。本研究的重点是反密码子茎环E内的修饰位点。coli tRNAPhe和双特异性修饰酶假尿苷合酶RluA的rRNA靶标。 具体而言，将讨论以下主题：（1）MiaA RNA配体的化学和酶促保护，以确定与MiaA蛋白接触的RNA上的位置，并监测结合诱导的RNA结构变化，（2）与MiaA复合的RNA配体的结构研究，以及（3）来自E. coli 23 S rRNA，RluA酶的“第二”靶标。这些研究的结果与理解蛋白质-RNA相互作用的特异性和亲和力以及修饰对RNA结构和动力学的影响及其与RNA功能（例如翻译）的关系直接相关。这些调查也将补充MiaA催化的异戊二烯化反应的机理研究。许多RNA分子与特定的蛋白质分子相互作用，或者被这些蛋白质修饰，或者作为蛋白质-RNA复合物执行独特的细胞功能。因此，了解编码单个蛋白质-RNA相互作用特异性的分子原理具有基本的生物学重要性。目前，已知的蛋白质-RNA识别的分子细节只有少数复合物，甚至更少的识别的一般原则已经出现。 本研究的总体目标是确定两种模式细菌蛋白质的蛋白质-RNA识别规则，并确定由其酶促作用引入的结构和稳定性效应。

Nikonowicz, Edward P.MCB 0078501The goal of this project is to understand key aspects of the post-transcriptional modification process of tRNA at the molecular level, including the basis of molecule specific RNA recognition by modification enzymes and the functional basis of modifications within RNA molecules. This research focuses on modification sites within the anticodon stem-loop E. coli tRNAPhe and the rRNA target of the dual specificity modification enzyme pseudouridine synthase RluA. Specifically, the following topics will be addressed: (1) chemical and enzymatic protection of MiaA RNA ligands to identify positions on the RNA that contact MiaA protein and to monitor binding induced RNA structure changes, (2) structural studies of an RNA ligand in complex with MiaA, and (3) structure determination of Helix-35 from E. coli 23S rRNA, the "second" target of the RluA enzyme. The results of these studies are directly relevant to understanding the specificity and affinity of protein-RNA interactions and the impact of modification on RNA structure and dynamics and its relationship to RNA function (e.g. translation). These investigations also will complement mechanistic studies of the prenylation reaction catalyzed by MiaA. Many RNA molecules interact with specific protein molecules either to be modified by those proteins or to perform unique cellular functions as a protein-RNA complex. Thus, it is of fundamental biological importance to learn the molecular principles that encode the specificity of individual protein-RNA interactions. At present, the molecular details of protein-RNA recognition are known for only a small number of complexes and even fewer general principles for recognition have emerged. The overall goal of this research is to determine the rules of protein-RNA recognition for two model bacterial proteins and to determine the structural and stability effects introduced by their enzymatic action.