SINGLE-SITE MUTATIONS IN THE C-TERMINAL DOMAIN OF BACTERIOPHAGE-LAMBDA CI-REPRESSOR ALTER COOPERATIVE INTERACTIONS BETWEEN DIMERS ADJACENTLY BOUND TO O-R

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 bacteriop hage lambda [Johnson, A. D., et al. (1981) Nature 294, 217-223; Brenow itz, M., et al. (1986) Methods Enzymol. 130, 132-181]. Quantitative mo deling has suggested that cooperativity is required for maintainence o f the lysogenic state and for the efficient switch from lysogenic to l ytic 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 m olecule [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 medi ating 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 3 2, 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 s ystem requires that the equilibrium dimerization constants and the int rinsic and cooperative free energies of binding be measured. In this w ork we evaluate the interaction of eight mutant repressors with O-R DN A: Gly147-->Asp (GD147), Pro158-->Thr (PT158), Glu188-->Lys (EK188), L ys192-->Asn (KN192), Tyr210-->His (YH210), Ser228-->Arg (SR228), and S er228-->Asn (SN228), each with an amino acid substitution in the C-ter minal domain, and Glu102-->Lys (EK102) where the substitution lies in the ''linker sequence'' between domains. Self-assembly properties of s ix of these mutant repressors are presented in the preceding paper (Bu rz et al., 1994). In this work, the binding of mutant cI repressors to O-R was examined using quantitative DNAse I footprinting. This techni que monitors individual site occupancy concurrent with binding at the other sites within a multisite complex. Simultaneous analysis of titra tion data for mutant repressors on wild-type and ''reduced valency'' O -R DNA shows that the intrinsic free energy of binding to individual o perator sites for the mutants is essentially unchanged relative to tha t of wild type, while the magnitudes of cooperative DNA binding intera ctions fall into three general classes: WT, EK102, and SN228, which ex hibit greater than similar to 2.5 kcal/mol of cooperative free energy; EK188 and SR228, which exhibit 1-2 kcal/mol of cooperativity; and GD1 47, KN192, and YH210, which are essentially devoid of cooperative bind ing free energy. The resultant deficiencies in cooperative interaction s support the proposal that the origins of cooperativity may reside wi thin the C-terminal domains. This detailed characterization of coopera tivity mutants with facilitate ongoing in vitro studies regarding the molecular mechanism of regulation and in vivo studies aimed at elucida ting the role of cooperativity in the life cycle of bacteriophage lamb da.