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.
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.
Rogozina
Anastasia
A
Max Planck Institute of Biochemistry, D82152 Martinsried bei Munchen, Germany.
Zaychikov
Evgeny
E
Buckle
Malcolm
M
Heumann
Hermann
H
Sclavi
Bianca
B
D017123:Bacteriophage T7 / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013696:Temperature; D014158:Transcription, Genetic
D011196:Potassium Permanganate; D004247:DNA; D012321:DNA-Directed RNA Polymerases