Rapid-Quench Mixing and Use of Fast Footprinting to Characterize DNA Opening in the Late Steps of Open Complex Formation at λPR by E. coli RNA Polymerase

Abstract

The precise mechanism by which multi-subunit, DNA-dependent RNA polymerases (RNAP) recognize promoter DNA and form stable, transcriptionally-competent open complexes (RPo) remains elusive. Chemical and enzymatic footprints of early intermediates and RPo provide detailed pictures of the DNA in these initial and final complexes in transcription initiation. We have recently discovered that the start site for transcription (+1) remains double-stranded when it is first loaded into the active site channel of E. coli RNAP at the λ phage PR promoter (Davis et al. PNAS, 2007). These experiments reveal unambiguously that opening of the transcription bubble occurs subsequently in the “jaws” of RNAP as RNAP actively destabilizes the duplex. To probe whether opening of ∼14 bp of DNA occurs in stages or is “all or none” we are using the perturbants urea and KCl to destabilize RPo and populate late transcription initiation intermediates. Analysis of the dissociation kinetics over a range of urea and KCl concentrations provides evidence for two transient intermediates in the DNA opening process. Using KMnO4 footprinting we are probing the extent of DNA opening in these intermediates under these conditions. Results from these novel rapid (millisecond) footprinting experiments and the implications for the mechanism of promoter opening will be presented.