DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37 degrees C and results in the accumulation of an off-pathway intermediate.

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO(4) and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts.

DNA polymerase beta reveals enhanced activity and processivity in reverse micelles.

Water is essential for the stability and functions of proteins and DNA. Reverse micelles are simple model systems where the structure and dynamics of water are controlled. We have estimated the size of complex reverse micelles by light scattering technique and examined the local microenvironment using fluorescein as molecular probe.

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter.

We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37 degrees C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the alpha-subunit C-terminal domain and binding of the sigma subunit to the -35 sequence.