Freedman L P, Luisi B F, Korszun Z R, Basavappa R, Sigler P B, Yamamoto K R
Department of Biochemistry & Biophysics, University of California, San Francisco 94143-0448.
Nature. 1988 Aug 11;334(6182):543-6. doi: 10.1038/334543a0.
The glucocorticoid receptor enhances or represses transcription by binding to specific DNA sequences termed glucocorticoid response elements, or GREs. Studies of cloned glucocorticoid receptors reveal that the protein is organized as functional domains, in an arrangement that appears to be common among members of the steroid receptor family. A segment near the centre of the gene specifies DNA binding activity in vitro and contains two sequence motifs similar to 'zinc fingers' found in Xenopus transcription factor IIIA (TFIIIA). Such sequence motifs have been identified in nucleic acid binding proteins from a wide range of organisms. Steroid receptor protein fingers are proposed to bind zinc through two pairs of conserved cysteine residues. We report here that a protein of relative molecular mass 19,000 (Mr = 19 K) encompassing the DNA-binding domain of the glucocorticoid receptor that has been overexpressed in Escherichia coli and purified to homogeneity reversibly ligates two Zn(II) or Cd(II) ions. We show that metal ions are required for specific DNA binding and proper folding. Using EXAFS (extended X-ray absorption fine structure) and visible light spectroscopies, we find that each Zn atom is coordinated in a tetrahedral arrangement by four cysteines.
糖皮质激素受体通过与特定的DNA序列（称为糖皮质激素反应元件或GREs）结合来增强或抑制转录。对克隆的糖皮质激素受体的研究表明，该蛋白质被组织成功能结构域，其排列方式在类固醇受体家族成员中似乎很常见。基因中心附近的一个片段在体外指定了DNA结合活性，并包含两个与非洲爪蟾转录因子IIIA（TFIIIA）中发现的“锌指”相似的序列基序。在来自多种生物体的核酸结合蛋白中已鉴定出此类序列基序。类固醇受体蛋白指被认为通过两对保守的半胱氨酸残基结合锌。我们在此报告，一种相对分子质量为19,000（Mr = 19 K）的蛋白质，它包含糖皮质激素受体的DNA结合结构域，已在大肠杆菌中过表达并纯化至同质，该蛋白质可逆地连接两个Zn(II)或Cd(II)离子。我们表明金属离子是特异性DNA结合和正确折叠所必需的。使用扩展X射线吸收精细结构（EXAFS）和可见光光谱学，我们发现每个Zn原子由四个半胱氨酸以四面体排列配位。
