FIG 3.

Vfr binds specifically to exlBA promoter in a cAMP-dependent manner. (A and B) The recombinant His₆-Vfr protein was incubated with 0.5 nM Cy5-labeled exlBAp probe (A) or the mutated exlBAp-mut probe (B) for 15 min before electrophoresis. Arrows indicate the positions of unbound free probes and Vfr-promoter probe complexes. The concentrations of Vfr used in the assay are also shown above each gel. (C) His₆-Vfr (50 nM) was incubated with 0.5 nM Cy5-exlBAp probe, and where indicated, a 200-fold excess of unlabeled wild-type or mutated probe was added to the reaction mixture prior to incubation. (D) His₆-Vfr (50 nM) was incubated with Cy5-exlBAp probe (0.5 nM) in the presence or absence of 20 μM cAMP in the binding buffer, as indicated. Note that in panels A, B, and C, cAMP (20 μM) was provided in the binding assay.