Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

The architecture of cellular RNA polymerases (RNAPs) dictates that transcription can begin only after promoter DNA bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. Formation of this transcriptionally competent "open" complex (RP.) by Escherichia coli RNAP at the lambda P-R promoter is greatly accelerated by DNA upstream of base pair -47(with respectto +1). Herewe report real-time hydroxyl radical (center dot OH) and potassium permanganate (KMnO4) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate (I-1) in RP degrees formation. center dot OH foot-prints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I-1 but not in RP degrees; downstream protection extends to approximately +20 in both complexes. KMnO4 footprinting detects solvent-accessible thymine bases in RP degrees, but not in I-1. We conclude that upstream DNA wraps more extensively on RNAP in I-1 than in RPO and that downstream DNA (-11 to +20) occupies the active-site channel in I-1 but is not yet melted. Mapping of the footprinting data onto available x-ray structures provides a detailed model of a kinetic intermediate in bacterial transcription initiation and suggests how transient contacts with upstream DNA in I-1 might rearrange the channel to favor entry of downstream duplex DNA.