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 an d P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resi stant manner. Binding of polymerase to this site, termed P3, occurs simulta neous to its binding at P1/P2. We have located this P3 site by DNase I foot printing. 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 tr anscription occurs from P3 in vitro. Primer extension analysis provides dir ect evidence that P3 is transcribed in vivo. The start site of transcriptio n has been mapped at -96 position relative to galP1. beta -galactosidase as says with different gal promoter constructs reveal that while P3 alone func tions as a weak in vivo promoter, it has a synergistic effect on transcript ion from the gal operon, since deletion of P3 or specifically mutating its -10 region result in a substantial reduction in the gal promoter activity.