Zilliacus J, Dahlman-Wright K, Wright A, Gustafsson J A, Carlstedt-Duke J
Department of Medical Nutrition, Huddinge University Hospital, Sweden.
J Biol Chem. 1991 Feb 15;266(5):3101-6.
Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.
在雌激素受体的DNA结合结构域中，少数氨基酸突变为糖皮质激素受体的相应序列，可在反式激活试验中改变受体的特异性（马德，S.，库马尔，V.，德韦尔内伊，H.，和尚邦，P.（1989年）《自然》338卷，271 - 274页）。我们在糖皮质激素受体DNA结合结构域的背景下进行了相应的反向突变，并使用凝胶迁移率变动分析研究了野生型和突变型纯化蛋白与回文糖皮质激素和雌激素反应元件以及中间序列元件的结合情况。我们在此表明，有两个氨基酸改变的蛋白以低但相等的亲和力结合糖皮质激素和雌激素反应元件，而有一个额外改变残基的蛋白对雌激素反应元件具有高亲和力，但对糖皮质激素反应元件仍保留相当大的亲和力。利用中间序列的结合位点，我们进一步表征了与DNA的相互作用。体外DNA结合结果在酵母体内的反式激活试验中得到了证实。最后，我们提出了一个可测试的模型，用于解释糖皮质激素受体DNA结合结构域识别其靶序列过程中涉及的氨基酸/碱基对相互作用。
