Investigating Mechanisms of RNA Polymerase II Transcription and Regulation Using Single-molecule Fluorescence

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

Intellectual Merit: The process of mRNA synthesis (transcription) is a principle control point for regulating gene expression during growth, development, and normal cellular metabolism. The first step in transcription is formation of a complex of proteins at the promoter of the DNA template. The goal of this project is to investigate the assembly mechanism, dynamics, and heterogeneity of human transcription factor/DNA complexes, and how these parameters contribute to transcriptional control. This research will take advantage of single-molecule fluorescence techniques, which have emerged as essential contributors to revealing the dynamic behavior, conformational states, and heterogeneity of biological complexes, thus providing unprecedented insight into their function. Specifically, studies will investigate human HMGB1, a biologically essential architectural protein that facilitates the formation of protein/DNA complexes important in transcriptional regulation by dynamically binding and bending DNA. The mechanism by which HMGB1 binds and distorts DNA, and how this activity facilitates DNA binding by the transcriptional activator protein p53 will be determined. The research will also investigate the parameters that define the assembly and activity of complexes containing human RNA polymerase II, general transcription factors, and promoter DNA. Fundamental questions about the macromolecular interactions that govern the onset of transcription will be answered. The results from the research will be integrated with previously published biochemical and cell-based studies to provide a comprehensive view of the mechanism by which HMGB1 functions and how RNA polymerase II complexes assemble and transcribe. Broader impacts: The future of science is intimately tied to integrating research and education. Graduate and undergraduate students will perform the research in this project, and significant effort will be made in training these students in experimental design, interpretation of data, creative thought, preparation of manuscripts, and oral presentations. Importantly, efforts will continue to be made to recruit underrepresented minority students into the laboratory. The project also contains an educational component. An experimental module for an undergraduate laboratory course will be developed and implemented. It will teach the relationship between fluorescence energy resonance transfer efficiency and distance between fluorophores in a discovery-based experiment emphasizing experimental design, application of acquired knowledge, data interpretation, and literature searches.