COOPERATIVE PROTEIN DNA INTERACTIONS - EFFECTS OF KCL ON LAMBDA-CI BINDING TO OR

The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites O(R) were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type O(R) and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, DELTA-G1, DELTA-G2, and DELTA-G3, and cooperative interactions, DELTA-G12 and DELTA-G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites O(R)1 and O(R)3 change in parallel over the entire range of [KCl]. The KCl dependencies at O(R)1 and O(R)3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency Of O(R)2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.