Use of Fast DNA Footprinting and Real-Time Fluorescence Spectroscopy to Characterize Novel, Biologically Revlevant Transcription Initiation Intermediates for E. Coli RNA Polymerase

Abstract

RNA polymerase (RNAP) is a complex molecular motor responsible for catalyzing the synthesis of RNA from DNA in a promoter sequence-dependent manner. Knowledge of the mechanism of transcription initiation is crucial to understanding the regulation of gene expression. However, the RNAP-DNA intermediates on the pathway to open complex formation during transcription initiation are extremely short-lived and thus have been very difficult to characterize using traditional structural methods. Here, we present work focused on developing rapid, solution-based methods that allow us to characterize RNAP-DNA structures in the millisecond time regime. These include: fast DNA footprinting (using hydroxyl radical and permanganate probes) to characterize the interaction of RNAP with the DNA backbone and the extent of thymine base unstacking; stopped-flow FRET experiments to monitor DNA bending in real time; and 2-aminopurine fluorescence experiments to characterize the kinetics of DNA opening. In a related project, we utilize thin layer chromatography and fluorescence spectroscopy to characterize the short (2, 3 and 4-mer) RNA products made by RNAP during repeated rounds of abortive cycling before the enzyme progresses to forming full-length RNA transcripts. These combinatorial approaches have allowed us to determine the timing of loading of duplex DNA into the RNAP active site cleft and the kinetic mechanism of large-scale RNAP conformation changes leading to initial RNA synthesis. These research activities have successfully developed new methods to characterize transient intermediates in solution and to begin to develop a detailed structural-mechanistic picture of transcription initiation.