Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy.
Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.
Ozers
M S
MS
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Hill
J J
JJ
Ervin
K
K
Wood
J R
JR
Nardulli
A M
AM
Royer
C A
CA
Gorski
J
J
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009994:Osmolar Concentration; D011960:Receptors, Estrogen / Q000378:metabolism*; D011994:Recombinant Proteins; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D004958:Estradiol; D004247:DNA