Figure 3.

Quantitation of OmpR binding to the F1-F1 fragment. A DNA fragment (F1-F1) containing a tandem duplication of the F1 site was labeled using [α-³²P]dATP. The labeled fragments (≈0.13 nM) were incubated with increasing amounts of OmpR in the absence (•) or presence (○) of the kinase (0.63 μM), and the resulting OmpR/DNA complexes were resolved on a nondenaturing gel containing 10% polyacrylamide (30:1; wt/wt, acrylamide to bisacrylamide) followed by autoradiography. The amount of free DNA and bound DNA in the different complexes was determined as described. In A, the fraction of free DNA was measured and then used to calculated the total fraction of bound DNA. The total percentage of bound DNA observed was then plotted as a function of OmpR concentration. In B, the fraction of the intermediate species (I), which corresponds to occupancy of only one site, was plotted as a function of OmpR concentration. The fractional maximum of species I was then determined and used to calculate the cooperativity factor (k_dI/k_dII), as described.