The proposed research will describe how steroid binding to its receptor alters the equilibrium binding of the calf uterine estrogen and progesterone receptors to DNA. The association constant for binding to DNA and the cooperativity of protein-DNA interactions will be measured in the absence and presence of steroid hormones. Association constants will be determined by using chromatography of each receptor over small DNA-Sepharose columns at a constant salt concentration. The degree of cooperativity will be measured using "salt back-titration" analysis. This is based on measuring the salt-dependence of protein-DNA binding at high concentrations of protein. Free receptor and receptor bound to DNA will be separated by sucrose density gradient centrifugation and quantified by liquid scintillation counting of tritiated steroid. By quantifying the equilibrium binding of the estrogen and progesterone receptors to DNA in the absence and presence of different ligands, these experiments will describe some of the conformational changes in steroid receptors which are induced by ligand binding. Estrogen and progesterone receptors are proteins which bind steroid hormones. Hormone binding to the receptor induces a conformational change in the receptor which, in turn, alters the binding of the receptor to specific DNA sequences. Binding of the receptor to DNA leads to alterations in the expression of specific genes. The proposed quantitative studies of the interaction between the estrogen and progesterone receptors and DNA, and how DNA-binding of the receptors is altered by steroid binding will increase our basic understanding of the mechanisms by which gene expression is regulated by steroid hormones.
