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Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter.

TitleReal-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter.
Publication TypeJournal Article
Year of Publication2005
AuthorsSclavi, B, Zaychikov, E, Rogozina, A, Walther, F, Buckle, M, Heumann, H
JournalProc Natl Acad Sci U S A
Volume102
Issue13
Pagination4706-11
Date Published2005 Mar 29
ISSN0027-8424
KeywordsDNA, DNA Footprinting, DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Kinetics, Models, Molecular, Promoter Regions, Genetic, Protein Binding, Protein Conformation, Synchrotrons
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.

DOI10.1073/pnas.0408218102
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID15738402
PubMed Central IDPMC555702