A Consensus Adenine at Position –11 of the Nontemplate Strand of Bacterial Promoter Is Important for Nucleation of Promoter Melting*

Numerous studies have suggested an important role of adenine at position –11 of the nontemplate strand of bacterial promoters for sequence-specific recognition of the –10 promoter element in single-stranded form. In this work, we attempted to identify a specific step in transcription initiation reaction that is most critically dependent on specific recognition of –11A. Mutating –11A in the context of a model promoter resulted in a profound decrease of the rate of heparin-resistant promoter complex formation and in a modest increase of the rate of heparin-resistant complex dissociation. The identity of nontemplate base at position –11 became relatively unimportant when the duplex in the vicinity of this position was destabilized by base pair mismatches. For promoters with a nonnative thymine at nontemplate position –11, we observed a remarkable correlation between the rate of heparin-resistant complex formation (or transcription activity) and the free energy of duplex stability in the vicinity of this residue, indicating that the replacement of –11A with a T affected a step in the reaction that involves local melting of DNA duplex. These data show that a promoter melting defect caused by a loss of RNA polymerase contact with –11A can be rescued by artificially induced local destabilization of the DNA duplex. These results are consistent with and support the idea that specific recognition of adenine at the nontemplate –11-position is important only for the initial nucleation of melting, which probably involves the flipping of this adenine out from the DNA duplex.