Nucleic Acids Research Advance Access originally published online on July 3, 2009
Nucleic Acids Research 2009 37(16):5390-5404; doi:10.1093/nar/gkp560
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Nucleic Acids Research, 2009, Vol. 37, No. 16 5390-5404
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37°C and results in the accumulation of an off-pathway intermediate
1Max Planck Institute of Biochemistry, Am Klopferspitz 18A, D82152 Martinsried bei Munchen, Germany and 2LBPA, UMR 8113 CNRS, ENS Cachan, 94235 Cachan, France
*To whom correspondence should be addressed. Tel/Fax: +33 1 47 40 76 77; Email: sclavi{at}lbpa.ens-cachan.fr
Received February 17, 2009. Revised June 17, 2009. Accepted June 17, 2009.
The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild-type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus –10 region. Finally, the comparison of the results obtained at 37°C with those at 20°C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.