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Escherichia coli RNA Polymerase Recognition of a ⁷⁰-Dependent Promoter Requiring a –35 DNA Element and an Extended –10 TGn Motif

India Hook-Barnard, Xanthia B. Johnson, and Deborah M. Hinton^*

Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8, Room 2A-13, Bethesda, Maryland 20892-0830

Received 14 June 2006/ Accepted 25 September 2006

Escherichia coli 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 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.

Published ahead of print on 29 September 2006.

Present address: My Sister's Place, Domestic Violence Program, Washington, D.C.