Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes.
Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.
Brodolin
K
K
Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia. Brodolin@img.ras.ru
Buckle
M
M
D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D005557:Formaldehyde / Q000737:chemistry*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D005557:Formaldehyde; D004247:DNA; D012321:DNA-Directed RNA Polymerases