A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter-like activity.

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position - 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (-103 to -108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at -96 position relative to galP1. beta-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its -10 region result in a substantial reduction in the gal promoter activity.

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