title: Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter.
abstract: We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37 degrees C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the alpha-subunit C-terminal domain and binding of the sigma subunit to the -35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the -10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the beta/beta' jaws, leading to a transcriptionally active complex.
authors.affiliation: Max Planck Institute of Biochemistry, Am Klopferspitz 18A, D82152 Martinsried bei München, Germany. scalvi@lbpa.ens-cachan.fr
authors.forename: Bianca
authors.initials: B
authors.lastname: Sclavi
authors.forename: Evgeny
authors.initials: E
authors.lastname: Zaychikov
authors.forename: Anastasia
authors.initials: A
authors.lastname: Rogozina
authors.forename: Ferdinand
authors.initials: F
authors.lastname: Walther
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.forename: Hermann
authors.initials: H
authors.lastname: Heumann
mesh_terms: D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017356:Synchrotrons
chemical_list: D029968:Escherichia coli Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases
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