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.
Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia. Brodolin@img.ras.ru
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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