RNA Polymerase: Mechanism of Action

East Carolina University, Greenville NC

Investigators

Abstract

RNA polymerase (RNAP) is the key enzyme in the process of gene expression. This project will address the mechanism of translocation of RNAP during elongation and the mechanism for transcription initiation. The mechanism of translocation is hotly debated. In the case of transcription initiation, it is the most regulated step in gene expression. However, the mechanism for initiation and the reasons for the greater rate of nucleotide incorporation during elongation relative to initiation are unresolved. Stopped-flow kinetic studies based on the intrinsic protein fluorescence of RNAP will be used to monitor conformational events in RNAP upon the binding of different ligands, quench-flow kinetic studies will be conducted to ascertain the overall rate of nucleotide incorporation and stopped-flow kinetic studies will be used to monitor PPi release after nucleotide incorporation. The results of these studies will provide a detailed picture of the mechanism for translocation during elongation involving conformational changes in RNAP associated with the various steps in the process. A comparison of corresponding rate constants obtained during initiation and elongation will allow identification of the step(s) that result in the differential rate of nucleotide incorporation during initiation and elongation. Moreover, the data will provide a detailed mechanism for nucleotide incorporation during initiation. The results obtained in these studies should provide new insight into the mechanism of action of RNAP. The broader impact of the proposed studies will be the exposure of a diverse group of young investigators to the scientific process in approaching studies in the area of basic science. The group will include college and medical students from underrepresented groups. Medical students will have an opportunity to conduct basic research in the summer after their first year in medical school. College students will be employed mainly in the summer months. All participants will contribute to the scientific studies and will be encouraged to try to develop their own ideas. They will use state-of-the-art equipment and will be exposed to a wide range of techniques. They will present their results in local and state meetings. Presentations at national meetings by the students will occur whenever feasible. This program will demonstrate to the young investigators that a career in science can be rewarding and fulfilling.