Fluorescence study of the multiple binding equilibria of the galactose repressor

Biochemistry
K WangL Brand

Abstract

A fluorescence assay has been developed to study the multiple linked equilibria which function in regulation of the Escherichia coli galactose operon. Fluorescein 5-isothiocyanate was attached to Amino-Modifier C6dT at different positions in an oligonucleotide containing the sequence for the OE site of the galactose operon. These fluorescently labeled oligonucleotides were used to study OEDNA-GalR-d-galactose interactions. The data were analyzed and fit to various models including the 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 parameters with those obtained in independent experiments were used to distinguish different models. Since the ternary complex is likely to exist under physiological conditions, our results suggest that obligatory dissociation of GalR from DNA may not be required for induction of the gal operon. Rather, induction may involve the formation of a ternary complex of OEDNA with GalR and D-galactose with a different conformation than the GalR-OEDNA binary repressor complex.

Citations

Apr 21, 2004·Journal of Molecular Biology·Michael F BaileyDavid P Millar
Feb 1, 2002·Nature Biotechnology·Tomasz Heyduk, Ewa Heyduk
Sep 11, 2020·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Mathieu FossépréMathieu Surin
May 24, 2008·Analytical Chemistry·Xiaoyang XuChad A Mirkin
Nov 1, 2005·EcoSal Plus·Christoph Mayer, Winfried Boos

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