Escherichia coliRNA Polymerase Recognition of a σ70-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif

Abstract

Escherichia coli σ⁷⁰-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 σ⁷⁰ −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.