Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA-binding domain.
We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Härd
T
T
Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
Dahlman
K
K
Carlstedt-Duke
J
J
Gustafsson
J A
JA
Rigler
R
R
D000494:Allosteric Regulation; D001483:Base Sequence; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011189:Potassium Chloride; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014443:Tyrosine
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D014443:Tyrosine; D011189:Potassium Chloride