Koszewski N J, Notides A C
Department of Biophysics and Environmental Health Sciences Center, University of Rochester School of Medicine and Dentistry, New York 14642.
Mol Endocrinol. 1991 Aug;5(8):1129-36. doi: 10.1210/mend-5-8-1129.
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.
虽然已鉴定出构成雌激素反应元件（ERE）共有DNA序列的核苷酸碱基，但尚不清楚ERE的糖磷酸骨架与雌激素受体（ER）之间静电接触的参与情况。此外，这些接触对序列特异性DNA结合的贡献尚未确定。因此，通过磷酸乙基化干扰研究了高度纯化的ER与源自鸡卵黄生成素（cVit）-II基因的ERE磷酸残基之间的相互作用。在使用含有完美ERE（相对于基因起始位点为-625；5'-GGTCAGCGTGACC）或不完美ERE（-353；5'-GGTCAACATAACC）的DNA片段进行的电泳凝胶迁移率变动分析中，明显可见特异性的ER-DNA复合物。磷酸乙基化干扰足迹鉴定出与完美ERE特异性结合所必需的磷酸残基有2倍对称排除，且有5'交错，表明ER二聚体的每个单体都结合在DNA的大沟中。不完美ERE的干扰足迹未检测到受体与非编码链上反应元件3'半段中的磷酸残基之间的相互作用。相比之下，完美ERE的相应足迹显示ER与DNA的磷酸骨架之间有强烈相互作用。因此，这些静电接触的缺失很可能是ER对不完美ERE结合亲和力降低的原因。这些结果表明，ER与其同源反应元件的糖磷酸骨架之间的特异性接触是DNA序列识别和高亲和力结合的一个重要方面。
