Fluorescence study of the multiple binding equilibria of the galactose repressor.
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.
Wang
K
K
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Rodgers
M E
ME
Toptygin
D
D
Munsen
V A
VA
Brand
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization; D005690:Galactose / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; C050993:Galactose repressor proteins; D015345:Oligonucleotide Probes; D012097:Repressor Proteins; D004247:DNA; D005690:Galactose