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 a nd cooperativity of the natural system. Domain self-assembly was studi ed 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 a nd Ackers, 1991a), indicating that different driving forces dominate t he respective assembly processes. The interactions of O-R With N-termi nal domains were noncooperative under all conditions studied. Binding at each site is accompanied by a negative enthalpy (large at site 1, s mall at sites 2 and 3). Identical values for salt release and proton a bsorption were found for the three sites. Comparisons with the analogo us thermodynamic parameters from our previous studies indicate that N- terminal domains exhibit different linkages to pH, KCl, and T from tho se of intact cI-O-R interactions. This implies that the domains do not act independently within the intact repressor. Since the linkage diff erences are dependent upon which site the proteins are binding, the C- terminal domain must play a role in repressor discrimination between s pecific sites.