RUI: Mechanism of Promoter Escape by E. coli RNA Polymerase

Mount Holyoke College, South Hadley MA

Investigators

Abstract

In a round of transcription, RNA polymerase goes through three phases -- initiation, elongation, and termination -- to synthesize a full-length transcript. Promoter escape, which constitutes the initiation-elongation transition, is a key step in regulating productive gene expression. Failure of RNA polymerase to negotiate this transition results in the repetitive synthesis of abortive RNA, a process known as abortive initiation. The extent of abortive initiation vs. promoter escape differs from promoter to promoter and is guided by the initial transcribed sequence (ITS) at each promoter. While previous research has led to the description of a general outline of the promoter escape process, current research will be directed at achieving a detailed mechanism of promoter escape. This goal will require investigation into the sequence-specific nature of promoter escape to understand how an ITS directs a unique abortive initiation-promoter escape program at individual promoters, and controls productive expression by governing the rate of escape and the extent of RNA polymerase partitioning. The proof of a promoter escape mechanism will also require structural characterization of initial transcribing complexes poised on the brink of escape. The methods to be employed in this research will include quantitative in vitro transcription analysis of wild type and mutant promoter templates, kinetic determination of rate and equilibrium constants to understand better the partitioning of transcribing complexes into productive or unproductive conformation, and footprinting techniques to characterize the static or dynamic structure of transcribing complexes. The combined approaches will shed light on how the ITS sequence directs RNA polymerase to relinquish its promoter contacts at specific positions and bring about the escape transition at a defined rate. All of the knowledge gained from studying E. coli RNA polymerase will be of relevance to understanding the promoter escape strategies by T7 RNA polymerase and eukaryotic Pol II. Broader Impacts: Being affiliated with a women's college, the principal investigator routinely works with many undergraduate students in the research and teaching laboratories. The research provides many self-contained, well-designed, and cutting-edge projects for rigorous and critical investigation. A broad impact of the research projects leads to the training of undergraduates, especially young women and of diverse ethnicity, preparing them for research participation in a scientific career.