Figure 8.

Characterization of UP-lac hybrid promoters in vitro. (A) EMSA. The indicated lac promoter derivatives were incubated with RNAP. Lane 5 shows the CRP-dependent P1 complex of the wild-type lac promoter formed in the presence of CRP and cAMP. Lanes 1 and 4 correspond to the P2 complex of the wild-type lac promoter. (B) Potassium permanganate footprinting of UP4- and UP8-lac promoters. The arrowheads indicate the permanganate-sensitive sites. The numbers represent positions relative to the transcription start site of P1. (C) Abortive initiation assay. The products of the abortive initiation reaction were analyzed by electrophoresis on a 20% polyacrylamide gel containing 8 M urea. Lane 1 represents the products derived from the wild-type lac promoter in the presence of CRP and cAMP. The arrowhead indicates the major product, which presumably corresponds to the pppApApUpU product synthesized at the lac P1 promoter.