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J. Bacteriol. doi:10.1128/JB.00853-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

E. coli RNA polymerase recognition of a ⁷⁰-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif

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

^* To whom correspondence should be addressed. Email: dhinton{at}helix.nih.gov.

	Abstract

E. coli ⁷⁰-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and -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, which 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 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 -11 to +4. We also demonstrate that transcription from P_minor incorporates nontemplated NTPs at the 5' end of the P_minor transcript, which results in an anomalous assignment for the start site when using primer extension analysis. 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.