Phosphate-sensitive binding of the estrogen receptor to its response elements.
Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference. Specific ER-DNA complexes were evident in electrophoretic gel mobility shift assays using DNA fragments containing either the perfect ERE (-625 relative to gene start site; 5'-GGTCAGCGTGACC) or the imperfect ERE (-353; 5'-GGTCAACATAACC). The phosphate ethylation interference footprint identified a 2-fold, symmetrical exclusion of phosphate residues essential for specific binding to the perfect ERE with a 5' stagger, indicating that each monomer of the ER dimer is bound in the major groove of the DNA. The interference footprint of the imperfect ERE did not detect interactions between the receptor and the phosphate residues in the 3' half of the response element on the noncoding strand. In contrast, the corresponding footprint of the perfect ERE displayed strong interactions between the ER and the phosphate backbone of the DNA. Consequently, the absence of these electrostatic contacts very likely accounts for the reduced binding affinity of the ER for the imperfect ERE. These results indicate that specific contacts between the ER and the sugar-phosphate backbone of its cognate response elements are an important aspect of DNA sequence recognition and high affinity binding.
Koszewski
N J
NJ
Department of Biophysics and Environmental Health Sciences Center, University of Rochester School of Medicine and Dentistry, New York 14642.
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D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004951:Esterification; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010710:Phosphates / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*; D014819:Vitellogenins / Q000235:genetics
D010710:Phosphates; D011960:Receptors, Estrogen; D014819:Vitellogenins; D004247:DNA