Ion effects on the lac repressor--operator equilibrium.
The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sensitive to the ionic environment. The dependence of the observed equilibrium constant on salt concentration is analyzed according to the binding theory of Record et al. [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]. The number of ionic interactions in repressor--operator complex is deduced from the slopes of the linear log-log plots. About 11 ionic interactions are formed between repressor and DNA phosphates at pH 7.4 and about 9 ionic interactions at pH 8.0, in reasonable agreement with previous estimates. A favorable nonelectrostatic binding free energy of about 9-12 kcal/mol is estimated from the extrapolated equilibrium constants at the 1 M standard state. The values are in good accord with recent results for the salt-independent binding of repressor core and operator DNA. The effects of pH on the repressor--operator interaction are small, and probably result from titration of functional groups in the DNA-binding site of the protein. For monovalent salts, the equilibrium constant is slightly dependent on cation type and highly dependent on anion type. At constant salt concentration, the equilibrium constant decreases about 10000-fold in the order CH3CO2- greater than or equal to F- greater than Cl- greater than Br- greater than NO3- greater than SCN- greater than I-. The wide range of accessible equilibrium constants provides a useful tool for in vitro studies of the repressor--operator interaction.
D000838:Anions / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D004279:DNA, Viral / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007763:Lac Operon / Q000187:drug effects*; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride; D012097:Repressor Proteins / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*
D000838:Anions; D002414:Cations, Monovalent; D004279:DNA, Viral; D012097:Repressor Proteins; D014157:Transcription Factors; D015636:Magnesium Chloride; D012965:Sodium Chloride; D008274:Magnesium