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,). RT, quenched from 37 degreesC to 14 degreesC isomerised to a closed, competitor resistant, complex (RPLT). 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 two melting around the transcription start site. (C) 2001 Academic Press.