Remodeling of the σ70 subunit non-template DNA strand contacts during the final step of transcription initiation

Transcription initiation in bacteria requires melting of similar to 13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the beta subunit lobe I (amino acid residues 186-433) initiates melting of the - 10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (+1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by cross-linking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the sigma(70) subunit and the non-template strand of the - 10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new sigma(70)-DNA contact as well as stable beta' and beta subunit contacts with the non-template DNA downstream of the -10 promoter element were established. In terms of the two-step (upstream initiation/ downstream propagation) model of promoter melting, our data suggest that beta lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (beta' clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between sigma(70) and the -10 promoter element that might facilitate promoter escape and sigma release. (c) 2005 Elsevier Ltd. All rights reserved.