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DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37 degrees C and results in the accumulation of an off-pathway intermediate.

TitleDNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37 degrees C and results in the accumulation of an off-pathway intermediate.
Publication TypeJournal Article
Year of Publication2009
AuthorsRogozina, A, Zaychikov, E, Buckle, M, Heumann, H, Sclavi, B
JournalNucleic Acids Res
Volume37
Issue16
Pagination5390-404
Date Published2009 Sep
ISSN1362-4962
KeywordsBacteriophage T7, DNA, DNA Footprinting, DNA-Directed RNA Polymerases, Kinetics, Nucleic Acid Denaturation, Potassium Permanganate, Promoter Regions, Genetic, Protein Conformation, Temperature, Transcription, Genetic
Abstract

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 KMnO(4) 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 degrees C with those at 20 degrees C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.

DOI10.1093/nar/gkp560
Alternate JournalNucleic Acids Res.
PubMed ID19578065
PubMed Central IDPMC2760793