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.
Heyduk
Ewa
E
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104, USA.
Kuznedelov
Konstantin
K
Severinov
Konstantin
K
Heyduk
Tomasz
T
D000225:Adenine / Q000737:chemistry*; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry; D007700:Kinetics; D017895:Manganese Compounds / Q000494:pharmacology; D008957:Models, Genetic; D009154:Mutation; D010087:Oxides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D013816:Thermodynamics; D013997:Time Factors
D017895:Manganese Compounds; D010087:Oxides; C048856:permanganic acid; D006493:Heparin; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D000225:Adenine