tailieunhanh - Glucocorticoid receptor binds cooperatively to adjacent recognition sites

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. | The EMBO Journal - 2263, 1989 Glucocorticoid receptor binds cooperatively recognition sites Wolfgang Schmid1, Uwe Strahle1, Gunther Schutz1, Jacky Schmitt2 and Henk Stunnenberg2 1Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, and2European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, FRG Communicated by 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 experinents. Direct measurement of the relative affinity and indirect determination by competition showed > 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. Key words: glucocorticoid receptor binding/cooperativity/ eukaryotic expression system/vaccinia virus Introduction Steroid hormone receptors are conditional transcription activator proteins (Yamamoto, 1985; Evans, 1988; Beato, 1989). Native receptors are inactive in the absence of their cognate hormones and bind with high affinity and specificity to their target sites only after the ligand is bound to the receptor, a step which was called 'activation' or 'transformation' (Jensen et al., 1968). This was confirmed by genomic footprinting studies which demonstrated that the hormone responsive element of the tyrosine .