Details

Autor(en) / Beteiligte

• Davis, Caroline A
• Bingman, Craig A
• Landick, Robert
• Record, M. Thomas Jr
• Saecker, Ruth M

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2007-05, Vol.104 (19), p.7833-7838

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2007

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 (RPo) by Escherichia coli RNAP at the λPR 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 (I₁) in RPo formation. ·OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I₁ but not in RPo; downstream protection extends to approximately +20 in both complexes. KMnO₄ footprinting detects solvent-accessible thymine bases in RPo, but not in I₁. We conclude that upstream DNA wraps more extensively on RNAP in I₁ than in RPo and that downstream DNA (-11 to +20) occupies the active-site channel in I₁ 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 I₁ might rearrange the channel to favor entry of downstream duplex DNA.