Remodeling of the sigma super(7) super(0) Subunit Non-template DNA Strand Contacts During the Final Step of Transcription Initiation

• Published in: Journal of molecular biology 2005-07, Vol.350 (5), p.930-937
• Main Authors: Brodolin, K, Zenkin, N, Severinov, K
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2005.05.048

Transcription initiation in bacteria requires melting of ~13bp 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 super(7) super(0) 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 super(7) super(0)-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 super(7) super(0) and the -10 promoter element that might facilitate promoter escape and sigma release.