title: Self-association and DNA binding of lambda cI repressor N-terminal domains reveal linkage between sequence-specific binding and the C-terminal cooperativity domain.
abstract: The effects of temperature, protons, and KCl on self-assembly and site-specific binding of lambda cI N-terminal domains with operator sites OR 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-OR 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 KCl. 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 & Ackers, 1991a), indicating that different driving forces dominate the respective assembly processes. The interactions of OR 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-OR 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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: D L
authors.initials: DL
authors.lastname: Bain
authors.forename: G K
authors.initials: GK
authors.lastname: Ackers
mesh_terms: D000494:Allosteric Regulation; D010582:Bacteriophage lambda / Q000737:chemistry; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D011189:Potassium Chloride; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D011189:Potassium Chloride