Regulation of Sigma 54-Dependent Transcription by Core Promoter Sequences: Role of −12 Region Nucleotides

The tetranucleotide core recognition sequence (TTGC) of the sigma 54 promoter −12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo and in vitro. Deregulated promoters were identified, implying that this core element can mediate the response to enhancer-binding proteins. These promoters had in common a substitution at position −12 (consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were identified. Their analysis indicated that positions −13 (consensus G) and −15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the −13 and −15 positions contribute to closed-complex formation, whereas the −12 position has a stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that the −12 region core contains specific subsequences that direct the diverse RNA polymerase interactions required both to produce RNA and to restrict this RNA synthesis in the absence of activation.