title: "Footprint" titrations yield valid thermodynamic isotherms.
abstract: A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.
authors.forename: M
authors.initials: M
authors.lastname: Brenowitz
authors.forename: D F
authors.initials: DF
authors.lastname: Senear
authors.forename: M A
authors.initials: MA
authors.lastname: Shea
authors.forename: G K
authors.initials: GK
authors.lastname: Ackers
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D011506:Proteins; D004247:DNA; D003851:Deoxyribonucleases