Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements.
Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.
Tully
D B
DB
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7545.
Cidlowski
J A
JA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid
D002352:Carrier Proteins; D004279:DNA, Viral; D009363:Neoplasm Proteins; D011965:Receptors, Glucocorticoid; D004247:DNA