Interaction of the Escherichia coli Gal Repressor Protein with Its DNA Operators in Vitro

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (O_E and O₁) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683–9687] has suggested the role of a regulatory “looped complex” mediated by the association of Gal repressor dimers bound at O_E and O_I. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 °C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, ΔG_I and ΔG_E, and a constant describing cooperativity, ΔG_IE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 °C was ΔG_IE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. ΔG_E and ΔG_I had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, ΔC_p, of-2.3 kcal mol^‒1 deg⁻¹. Mobility-shift titration experiments conducted at 20 and 0 °C yielded values for ΔG_IE that were consistent with those resolved from the footprint analysis. Unique values of ΔG_IE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that ΔG_E = ΔG_I: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of O_E^‒ and O_I^‒ operators.