5390–5404 Nucleic Acids Research, 2009, Vol. 37, No. 16 Published online 3 July 2009 doi:10.1093/nar/gkp560 DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 378C and results in the accumulation of an off-pathway intermediate Anastasia Rogozina 1 , Evgeny Zaychikov 1 , Malcolm Buckle 2 , Hermann Heumann 1 and Bianca Sclavi 2, * 1 Max Planck Institute of Biochemistry, Am Klopferspitz 18A, D82152 Martinsried bei Munchen, Germany and 2 LBPA, UMR 8113 CNRS, ENS Cachan, 94235 Cachan, France Received February 17, 2009; Revised and Accepted June 17, 2009 ABSTRACT The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conforma- tional changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO 4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these com- plexes 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 378C with those at 208C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex. INTRODUCTION The binding of RNA polymerase to promoter DNA is a key step in the regulation of gene expression. This process has been described as a multi-step pathway that begins with the formation of a competitor-sensitive ‘closed com- plex’, followed by isomerization into a competitor-resis- tant complex. This isomerization process is distinguished by an initial phase during which nucleation of DNA melting takes place and the promoter DNA becomes completely protected from cleavage by DNaseI or hydro- xyl radicals (1–4), followed by a second step involving the complete melting of the transcription bubble and the formation of a transcriptionally active complex, also known as the ‘open complex’ (5). A series of conforma- tional changes in both the protein and the DNA are necessary to obtain the active complex, and each of these presents a possible target for both regulation and for subtle modulation during evolution (6–8). A precise description of the pathway leading to the formation of a transcriptionally active complex is thus necessary in order to understand how regulatory mechanisms control RNA polymerase activity at the promoter. At some strong promoters such as the A1 promoter of T7 phage (9,10) the Escherichia coli RNA polymerase holoenzyme (RNAP, made of six subunits, 2 a, b, b 0 , ! and s) is able to recognize and bind specifically to the promoter, melt the DNA and initiate transcription in the absence of additional transcription factors. The study of binding of RNAP to these promoters has been used to define the sequence of events for this process as described above (3,11–14). The T7A1 promoter’s wild-type (wt) sequence is characterized by the presence of an AT-rich UP element extending to –71, a near consensus –35 sequence (TTGACT instead of TTGACA) and a non- consensus –10 sequence (GATACT instead of TATAAT). The description of structural intermediates in the process of macromolecular recognition remains a chal- lenge due to the presence of short-lived, unstable com- plexes. We previously characterized the structural kinetics of binding of RNAP to the T7A1 promoter using time-resolved X-ray footprinting (14). The tens of millisecond temporal resolution of this technique allows the characterization of different intermediates not only by their extent of protection but also by their kinetic *To whom correspondence should be addressed. Tel/Fax: +33 1 47 40 76 77; Email: sclavi@lbpa.ens-cachan.fr ß 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.