SELF-ASSOCIATION AND DNA-BINDING OF LAMBDA CI REPRESSOR N-TERMINAL DOMAINS REVEAL LINKAGE BETWEEN SEQUENCE-SPECIFIC BINDING AND THE C-TERMINAL COOPERATIVITY DOMAIN

The effects of temperature, protons, and KCl on self-assembly and site-specific binding of lambda cI N-terminal domains with operator sites O-R were studied to assess the roles of these domains in DNA binding and cooperativity of the natural system. Domain self-assembly was studied using sedimentation equilibrium while domain-O-R interactions were analyzed by quantitative DNase footprint titration. The self-assembly reactions were modeled best as a monomer-dimer-tetramer stoichiometry. Compared with intact cI, the monomer-dimer assembly is energetically weak and is largely independent of pH and KCI. The van't Hoff enthalpy of dimerization was found to be large and positive (+10.8 kcal/mol): in sharp contrast to that of intact cI (i.e., -16.1 kcal/mol; Koblan and Ackers, 1991a), indicating that different driving forces dominate the respective assembly processes. The interactions of O-R With N-terminal domains were noncooperative under all conditions studied. Binding at each site is accompanied by a negative enthalpy (large at site 1, small at sites 2 and 3). Identical values for salt release and proton absorption were found for the three sites. Comparisons with the analogous thermodynamic parameters from our previous studies indicate that N-terminal domains exhibit different linkages to pH, KCl, and T from those of intact cI-O-R interactions. This implies that the domains do not act independently within the intact repressor. Since the linkage differences are dependent upon which site the proteins are binding, the C-terminal domain must play a role in repressor discrimination between specific sites.