FLUORESCENCE STUDY OF THE MULTIPLE BINDING EQUILIBRIA OF THE GALACTOSE REPRESSOR

A fluorescence assay has been developed to study the multiple linked e quilibria which function in regulation of the Escherichia coli galacto se operon, Fluorescein 5-isothiocyanate was attached to Amino-Modifier C6dT at different positions in an oligonucleotide containing the sequ ence for the O-E Site Of the galactose operon. These fluorescently lab eled oligonucleotides were used to study O(E)DNA-GalR-D-galactose inte ractions, The data were analyzed and fit to various models including t he classical competitive binding model as well as models involving the formation of a ternary DNA-repressor-inducer complex. Examination of the reduced chi-square of the various fits and comparison of fitted pa rameters with those obtained in independent experiments were used to d istinguish different models. Since the ternary complex is likely to ex ist under physiological conditions, our results suggest that obligator y dissociation of GalR from DNA may not be required for induction of t he gal operon. Rather, induction may involve the formation of a ternar y complex of O(E)DNA with GalR and D-galactose with a different confor mation than the GalR-O(E)DNA binary repressor complex.