Schmid W, Strähle U, Schütz G, Schmitt J, Stunnenberg H
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg, FRG.
EMBO J. 1989 Aug;8(8):2257-63. doi: 10.1002/j.1460-2075.1989.tb08350.x.
In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.
为了确定由多个糖皮质激素反应元件（GRE）介导的协同诱导机制，通过凝胶阻滞、硝酸纤维素滤膜结合及足迹实验分析了糖皮质激素受体对单个或重复GRE的亲和力。直接测量相对亲和力及通过竞争进行间接测定表明，与单个元件相比，糖皮质激素受体对重复GRE的亲和力高出10倍以上。使用位于DNA螺旋同一侧的两个紧密间隔的GRE可获得GRE-受体复合物的最大稳定性。增加GRE之间的距离或改变其螺旋位置会显著增加受体的解离速率。DNase I足迹实验表明，除了GRE区域受到保护外，未受保护的中间DNA区域的模式也发生了改变，这表明与相邻GRE结合的受体使DNA螺旋结构发生了改变。
