Dynamic regulation of initiation and pausing of transcription by human RNA polymerase II

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The human genome contains about 3 billion base pairs of DNA that provide essential instructions for cells to respond to developmental or environmental cues. A key step in the response is transcribing the information in DNA to intermediary RNA molecules that supply another layer of information. Transcribing DNA to RNA is a highly regulated process, but exactly how it is controlled remains an enigma. The goal of this project is to decipher this problem by bringing together an experienced team of investigators to take a multi-faceted approach to catalyze discovery in new ways. The research plan will integrate educational and outreach programs at University of Colorado-Boulder and University of California-Los Angeles to expose high school, college students, and postdoctoral scholars to high-impact collaborative research. The tools, strategies, and methodologies to be implemented will be applicable to the study of other biological processes and molecular machines.The questions addressed in this project are fundamentally important for understanding the molecular control of eukaryotic transcription. The human RNA polymerase II transcription machinery will be studied in detail using cutting-edge methods developed initially through analysis of bacterial RNA polymerase. Highly regulated stages in gene expression, including transcription initiation and pausing, will be the focus. This collaborative project will merge solution-based single-molecule fluorescence techniques with detailed functional assays enabled by biochemical reconstitution of the entire 4.5 MDa human transcription apparatus. Two key questions will be addressed: How is RNA polymerase II transcription dynamically regulated by specific protein factors? What role does RNA polymerase II backtracking play in regulating initiation and pausing? An important aspect of the work will be to test and validate mechanistic findings with in vitro and cell-based approaches.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.