The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.
The glucocorticoid receptor protein, in association with cognate hormonal ligands, binds with high affinity to specific DNA sequences termed glucocorticoid response elements (GREs) which can function as hormone-dependent transcriptional enhancers; thus, the receptor is a regulable enhancer-activating protein. We have constructed cell lines expressing different levels of glucocorticoid receptor, and demonstrate that the extent of a structural alteration in the chromatin at a characterized GRE, as well as the magnitude of several transcriptional responses elicited by the receptor, are roughly proportional to the number of receptor molecules per cell. Thus, for three independent glucocorticoid-responsive transcription units examined in our HTC-derived cell lines, the receptor appears to be a primary regulatory factor. Moreover, the results suggest that other cellular factors required for the assembly and function of GREs and transcription initiation complexes must be produced in excess relative to their levels of utilization at normal receptor concentrations.
Transcriptional enhancement is a general mechanism for regulation of gene expression in which particular proteins bound to specific DNA sequences stimulate the efficiency of initiation from linked promoters. One such protein, the glucocorticoid receptor, mediates enhancement in a glucocorticoid hormone-dependent manner. In this study, a region of the 795-amino acid rat glucocorticoid receptor that is active in transcriptional enhancement was identified. The active region was defined by expressing various receptor deletion mutants in stably and transiently transfected cells and examining the regulated transcription of hormone-responsive genes. Mutant receptors lacking as many as 439 amino-terminal amino acids retained activity, as did those with as many as 270 carboxyl-terminal amino acids deleted. This suggests that the 86-amino acid segment between the most extensive terminal deletions, which also includes sequences required for specific DNA binding in vitro, is sufficient for enhancer activation. In fact, a 150-amino acid receptor fragment that encompasses this segment mediates constitutive enhancement.
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