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

William Marsh Rice University, Houston TX

Investigators

Abstract

Nikonowicz, Edward P. MCB 0078501 The 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.