FIG. 4.

Stoichiometry of PhoP-DNA interactions. (A) Gel filtration chromatography was performed with ∼20 μg of purified PhoP. The inset shows a calibration curve of protein standards generated to determine approximate molecular mass of PhoP. Elution time of PhoP is indicated by an upward arrow. (B) PhoP-DNA complexes were resolved on a series of native polyacrylamide gels alongside nondenatured protein molecular mass standards, as described in Materials and methods. The retardation coefficient (K_r) values for protein standards were defined by Ferguson analysis and plotted as a function of molecular mass. Interpolation of −K_r (solid lines) indicated a molecular mass of ∼99.2 kDa for the PhoP-DNA complex. (C) DR1,2 comprising two 9-bp direct repeat motifs in tandem binds two molecules of PhoP. For the competitive EMSA experiment, ∼20 nM end-labeled DR1,2 DNA was incubated in binding reactions with recombinant forms of PhoP. Binding reactions contained no protein (lane 1), 4 μM PhoP alone (lane 2), 4 μM GST-PhoP alone (lane 13), and mixtures of indicated concentrations of PhoP and GST-PhoP (lanes 3 to 12). Protein-DNA complexes were analyzed as described for Fig. 1. HH, two molecules of PhoP; GG, two molecules of GST-PhoP; HG, one molecule each of PhoP and GST-PhoP bound to end-labeled DR1,2.