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.