Characterization of the Assembly and Function of E. Coli RNA Polymerase-Promoter DNA Open Complexes in Transcription Initiation

Abstract

Transcription of RNA is a highly regulated process, as it is a central component of cellular gene expression. Differences in biochemical rate constants and ligand affinities (promoter DNA, NTP, protein regulators) during isomerization steps of RNA polymerase (RNAP) prior to the transition from initiation to elongation are key regulators of gene expression and overall cellular activity. In order to develop predictive models for regulation of transcription initiation, quantitative characterization of the isomerization steps of open complex formation after recruitment of RNAP to promoter DNA are required. However, the transient nature (1 ms - 1s) of these intermediates has historically precluded characterization of the biologically relevant closed and open intermediates. We have developed methods to obtain near homogenous populations of two of these transient intermediates using high concentrations of RNAP and solute/salt upshifts that allows for the detection of differences in their structures and biochemical activity. We now propose to use bulk real-time fluorescence measurements to monitor DNA bending and to characterize the kinetics of DNA opening, to define the large-scale conformational changes that convert the initial closed complex to the open complex and to investigate the functional roles of the different open intermediates. This structural information will be used to gain insight into the type and extent of conformation changes in the RNAP machinery and to make testable predictions about the function and regulation of initation intermediates.