Kinetics And Mechanism Of Formation And Stabilization Of The Rna Polymerase-Promoter Open Compex

Specific binding of E. coli RNA polymerase holoenzyme (RNAP; α2ββ’σ70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the −10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Forster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061).