Intellectual Merit: The expression of information coded in cellular genomes requires DNA to be transcribed into RNA by RNA polymerase (RNAP). Thus, the initiation of transcription plays an essential role in the establishment and regulation of cellular phenotypes in all organisms from bacteria to man. This process consists of several steps including recruitment of RNAP to DNA promoter sequences, melting of double-stranded DNA, determination of a transcription start-site, initiation of NTP hydrolysis by RNAP, and transition into processive transcription elongation (promoter escape). Eukaryotic TATA-dependent promoters are conserved from yeast to man and direct the assembly of a pre-initiation complex that minimally consists of RNAP II, TATA-box binding protein, TFIIB, TFIIE, TFIIF, and TFIIH. While structural and biochemical approaches have resulted in a robust model of how initiation proceeds, many mechanistic details of eukaryotic transcription initiation remain unclear. This project aims to determine the kinetics of DNA structural transitions induced by pre-initiation complex formation using factors from Saccharomyces cerevisiae and single-molecule biophysical approaches. These techniques allow one to monitor individual active molecular complexes in real-time and determine both the nature of intermediate DNA structures and the rates at which the system moves between them. More specifically, the focus of this work is to determine the mechanism of this central molecular process by determining how the PIC manipulates promoter DNA during (1) DNA binding, (2) open complex formation, (3) start-site scanning, and (4) promoter escape.
Broader Impacts: This project has a direct impact on the training of postdoctoral researchers and graduate students for careers in research or science education. The single-molecule biophysical techniques used in the lab provide opportunities for graduate, undergraduate and high school students to gain experience in a unique biophysical methodology. Specifically, undergraduate students from the departments of physics and biomedical engineering work in the lab gaining exposure to these approaches in addition to molecular biology and biochemistry techniques. Additionally, undergraduate involvement also provides opportunities for postdocs and graduate students to gain experience as mentors. This first-hand experience allows them to more realistically consider career paths in science education at all levels. Participants include students and teachers from the Young Scientist Program at Washington University, which brings high school seniors and educators into the laboratory for summer research experiences. Conveniently, as transcription initiation represents a critical node in the central dogma of molecular biology, students at all levels are exposed to a wide range of concepts and experimental techniques involving DNA, RNA, and protein.
Co-funded by the Genetic Mechanisms Cluster in Molecular and Cellular Biosciences and by the Experimental Program to Stimulate Competitive Research.
