Comparison of operator-specific and nonspecific DNA binding of the lambda cI repressor: [KCl] and pH effects

The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.