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

Departments of *Biochemistry,
^§Bacteriology, and
^¶Chemistry and
^†Center for Eukaryotic Structural Genomics, University of Wisconsin, Madison, WI 53706

Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, and approved March 19, 2007 (received for review November 7, 2006)

Abstract

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 λP_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 (I₁) in RP_o formation. ·OH footprints reveal that the DNA backbone from −71 to −81 is engulfed by RNAP in I₁ 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 I₁. We conclude that upstream DNA wraps more extensively on RNAP in I₁ than in RP_o 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.

Footnotes

^‖To whom correspondence may be addressed at:
University of Wisconsin, 433 Babcock Drive, Madison, WI 53706.
E-mail: record{at}biochem.wisc.edu or rmsaecker{at}wisc.edu
Author contributions: C.A.D., M.T.R., and R.M.S. designed research; C.A.D., C.A.B., M.T.R., and R.M.S. performed research; R.L. contributed new reagents/analytic tools; C.A.D., C.A.B., M.T.R., and R.M.S. analyzed data; and C.A.D., C.A.B., R.L., M.T.R., and R.M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609888104/DC1.
Abbreviations:

RNAP,

RNA polymerase;

RPo,

open complex;

I1,

first kinetically significant intermediate;

CTD,

C-terminal domain;

NTD,

N-terminal domain.