Quantitative analysis of the glucocorticoid receptor-DNA interaction at the mouse mammary tumor virus glucocorticoid response element.
Purified glucocorticoid receptor (GR) from rat liver was used for a quantitative analysis of the protein-DNA interaction at specific GR-binding segments within the 5'-long terminal repeat of the mouse mammary tumor virus. A truncated receptor was generated and used to demonstrate formation of heterodimeric GR, which furthermore was shown to be in rapid equilibrium with receptor-monomer. The relative affinity for GR binding to specific GR sites versus random calf thymus DNA was approximately 2 x 10(3). At equilibrium a free GR concentration of 3 x 10(-10) M was required for half-maximal saturation of the two functionally important DNA sites within the mouse mammary tumor virus 5'-long terminal repeat. Although these two DNA segments act synergistically in mediating hormonal response, we did not detect cooperative GR binding to these regions in vitro. However, GR bound cooperatively within the downstream binding region. Similarly, GR was unable to facilitate factor binding to a neighboring nuclear factor 1 site, another essential element in the promoter. In contrast, nuclear factor 1 binding was inhibited slightly by GR.
Perlmann
T
T
Department of Molecular Genetics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Eriksson
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P
Wrange
O
O
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus; D003432:Cross-Linking Reagents; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003850:Deoxyribonuclease I; D008099:Liver / Q000378:metabolism*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D008969:Molecular Sequence Data; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013388:Succinimides; D014222:Triamcinolone Acetonide / Q000378:metabolism
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D011965:Receptors, Glucocorticoid; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D003850:Deoxyribonuclease I; D014222:Triamcinolone Acetonide