Figure 6. Interaction of OhrR with the promoter of ohrA and ohrR, as determined by EMSA.

Labeled probes containing the promoter region of ohrA (A) and ohrR (B) were incubated with increasing concentrations of purified OhrR protein, as indicated, and the mixture was separated in a polyacrylamide non-denaturing gel. Competition assays (right panels) using 250 nM OhrR were performed in the presence of 30-fold excess of unlabeled fragments of the same region (S) or the ohrR coding region (N) as competitors. (C and D) Effects of oxidants and DTT on OhrR binding. EMSA assays using OhrR with the ohrA promoter (C) or ohrR promoter (D). ³²P-Labeled probes were incubated with 250 nM purified OhrR. Either 0.3 mM tert-butyl hydroperoxide (tBOOH), 0.3 mM cumene hydroperoxide (CuOOH) or 1 mM H₂O₂ was added to the binding reaction and incubated for 10 min at 25°C. When indicated, DTT was then added into the reactions, and incubation continued at 25°C for 15 min.