Escherichia coli RNA polymerase recognition of a σ70-dependent promoter requiring a-35 DNA element and an extended-10 TGn motif

Escherichia coli sigma(70)-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and the -35 sequences or as extended -10 promoters (TGn/-10 promoters), which have the TGn motif and an excellent match to the -10 consensus sequence. We report here an investigation of a promoter, P-minor, that has a nearly perfect match to the -35 sequence and has the TGn motif. However, P-minor contains an extremely poor sigma(70) -10 element. We demonstrate that P-minor. is active both in vivo and in vitro and that mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P-minor the DNA is single stranded from positions -11 to +4. We also demonstrate that transcription from P-minor incorporates nontemplated ribonucleoside triphosphates at the 5' end of the P-minor transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P-minor represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.