MODULATION OF THE STABILITY OF A LAC REPRESSOR MEDIATED LOOPED COMPLEX BY TEMPERATURE AND IONS - ALLOSTERIC REGULATION BY CHLORIDE

The lactose repressor of Escherichia coli (LacI) associates to a biden tate tetramer in solution and can simultaneously bind two operators to form a protein-mediated ''looped complex''. Studies have been conduct ed of the binding of LacI to two operators separated by approximately 11 helical turns of DNA. Quantitative DNase I footprint titration anal ysis of the stability of the LacI-mediated looped complex reveals that the Gibbs free energy of cyclization (DELTAG-degrees j) of the looped complex of 11.7 +/- 0.4 kcal/mol is invariant with temperature. van't Hoff analysis reveals a large and positive enthalpy of cyclization (D ELTAH-degrees = 12.3 +/- 2.4 kcal/mol) and an entropy that is small an d positive (DELTAS-degrees = 2.2 cal/deg). Quantitative DNase I footpr int titration and kinetic dissociation studies were also conducted as a function of counter-ion type and concentration. Increasing concentra tions of KCl or potassium glutamate destabilize the looped complex, a result completely accounted for by increases in the intrinsic DNA-bind ing free energies. While the value of DELTAG-degrees j is invariant wi th ion concentration, chloride is a positive regulator. The value of D ELTAG-degrees j decreases by 1.5 kcal/mol upon substitution of chlorid e for glutamate. Measurements of DELTAG-degrees j conducted as a funct ion of chloride concentration at constant ionic strength reveal that a pproximately one chloride ion per tetramer is bound upon looped comple x formation. These results demonstrate specific allosteric regulation of the formation of the LacI-mediated looped complex by a mechanism di stinct from the regulation of the constituent protein-DNA interactions .