Remodeling of the σ70 Subunit Non-template DNA Strand Contacts During the Final Step of Transcription Initiation

Transcription initiation in bacteria requires melting of ∼13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the β 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 σ70 subunit and the non-template strand of the −10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new σ70-DNA contact as well as stable β′ and β 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 β lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (β′ clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between σ70 and the −10 promoter element that might facilitate promoter escape and σ release.