Wild-type cI repressor dimers bind with 2.5-3 kcal/mol of cooperative free energy to the tripartite right operator region (O-R) of bacteriophage lambda [Johnson, A. D., et al. (1981) Nature 294, 217-223; Brenowitz, M., et al. (1986) Methods Enzymol. 130, 132-181]. Quantitative modeling has suggested that cooperativity is required for maintainence of the lysogenic state and for the efficient switch from lysogenic to lytic growth [Ackers, G. K., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1129-1133; Shea, M. A., & Ackers, G. K. (1985) J. Mol. Biol. 181, 211-230]. Cooperativity and self-association are thought to involve protein-protein contacts between C-terminal domains of the repressor molecule [Pabo, C. O., et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1608-1612]. To address the importance of the C-terminal domain in mediating the cooperativity exhibited by lambda cI repressor, a number of single-site mutant candidates were screened for possible deficiencies in cooperative interactions [Beckett, D., et al. (1993) Biochemistry 32, 9073-9079; Burz, D. S., et al. (1994) Biochemistry 33, 8399-8405]. Since repressor dimerization and binding to operator sites are coupled processes, elucidation of the energetic basis of regulation in this system requires that the equilibrium dimerization constants and the intrinsic and cooperative free energies of binding be measured. In this work we evaluate the interaction of eight mutant repressors with O-R DNA: Gly147-->Asp (GD147), Pro158-->Thr (PT158), Glu188-->Lys (EK188), Lys192-->Asn (KN192), Tyr210-->His (YH210), Ser228-->Arg (SR228), and Ser228-->Asn (SN228), each with an amino acid substitution in the C-terminal domain, and Glu102-->Lys (EK102) where the substitution lies in the ''linker sequence'' between domains. Self-assembly properties of six of these mutant repressors are presented in the preceding paper (Burz et al., 1994). In this work, the binding of mutant cI repressors to O-R was examined using quantitative DNAse I footprinting. This technique monitors individual site occupancy concurrent with binding at the other sites within a multisite complex. Simultaneous analysis of titration data for mutant repressors on wild-type and ''reduced valency'' O-R DNA shows that the intrinsic free energy of binding to individual operator sites for the mutants is essentially unchanged relative to that of wild type, while the magnitudes of cooperative DNA binding interactions fall into three general classes: WT, EK102, and SN228, which exhibit greater than similar to 2.5 kcal/mol of cooperative free energy; EK188 and SR228, which exhibit 1-2 kcal/mol of cooperativity; and GD147, KN192, and YH210, which are essentially devoid of cooperative binding free energy. The resultant deficiencies in cooperative interactions support the proposal that the origins of cooperativity may reside within the C-terminal domains. This detailed characterization of cooperativity mutants with facilitate ongoing in vitro studies regarding the molecular mechanism of regulation and in vivo studies aimed at elucidating the role of cooperativity in the life cycle of bacteriophage lambda.
