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(o)) by Escherichia coli RNAP at the lambdaP(R) promoter is greatly accelerated by DNA upstream of base pair -47 (with respect to +1). Here we report real-time hydroxyl radical (*OH) and potassium permanganate (KMnO4) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate (I(1)) in RP(o) formation. .OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I(1) but not in RP(o); downstream protection extends to approximately +20 in both complexes. KMnO4 footprinting detects solvent-accessible thymine bases in RP(o), but not in I(1). We conclude that upstream DNA wraps more extensively on RNAP in I(1) than in RP(o) 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.
Davis
Caroline A
CA
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Bingman
Craig A
CA
Landick
Robert
R
Record
M Thomas
MT
Saecker
Ruth M
RM
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014158:Transcription, Genetic
D011196:Potassium Permanganate; D004247:DNA; D012321:DNA-Directed RNA Polymerases