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

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。本研究主要针对大肠杆菌抗密码子茎环tRNAPhe的修饰位点和双特异性修饰酶伪尿嘧啶合成酶RluA的rRNA靶点进行研究。具体而言，将讨论以下主题：(1)MiaA RNA配体的化学和酶保护，以确定与MiaA蛋白接触的RNA位置并监测结合诱导的RNA结构变化；(2)与MiaA复合物的RNA配体的结构研究；(3)从大肠杆菌23S rRNA （RluA酶的“第二”靶标）中确定Helix-35的结构。这些研究结果直接关系到理解蛋白质-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（如翻译）的关系。这些研究也将补充由MiaA催化的戊烯酰化反应的机理研究。许多RNA分子与特定的蛋白质分子相互作用，要么被这些蛋白质修饰，要么作为蛋白质-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重要的生物学意义。目前，蛋白质- rna识别的分子细节仅为少数复合物所知，而识别的一般原理则更少。本研究的总体目标是确定两种模式细菌蛋白的蛋白质- rna识别规则，并确定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.