K. Brodolin et M. Buckle, Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes, J MOL BIOL, 307(1), 2001, pp. 25-30
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, comp
lex (RPLT). We observed that contacts of the beta ' and sigma subunits with
the positions -3, -5 of the non-template DNA strand disappeared very quick
ly during the first 30 seconds after the temperature downshift. The re-anne
aling 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 trans
cription bubble was slower and completed within five minutes. The results s
upport a two-step model of promoter melting and suggest that conformational
changes in the RNA polymerase occur concurrently with two melting around t
he transcription start site. (C) 2001 Academic Press.