Properties of a high-affinity DNA binding site for estrogen receptor

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.