On the Specificity of the Binding of the Estradiol Receptor Protein to Deoxyribonucleic Acid

Abstract Steroid hormones appear to regulate gene expression in target tissues by binding tightly to a cytoplasmic receptor protein, thereby causing this protein to increase its affinity for regulatory sites on the genome. In this study, we have measured the hormone-dependent binding of estradiol receptor protein to a variety of nucleic acids and nucleoproteins, using salt conditions in the physiological range. Sedimentation partition chromatography, a new method for quantitating interactions between 2 macromolecules, allows a constant concentration of nucleic acid to be sedimented through a narrow zone containing the receptor. We use this method to estimate equilibrium dissociation constants, and find that the form of the estradiol receptor which sediments at 5 S binds to DNA at least 15-fold more tightly than does the 4 S form. Most strikingly, the 5 S receptor binds equally well (K = 300–400 µg per ml) to the following double-stranded DNAs: heterologous or homologous mammalian DNA, bacterial DNA, and polydeoxyadenylate-deoxythymidylate copolymer (a synthetic DNA); in contrast, no interaction was detected with double-stranded reovirus RNA. No significant binding of the 5 S receptor to either DNA or uterine chromatin was found when tested under these salt conditions by either gel permeation chromatography or standard sucrose gradient co-sedimentation assays. Both of these techniques are able to detect only interactions of much higher affinity than those observable by sedimentation partition chromatography or DNA-cellulose chromatography (Yamamoto, K., and Alberts, B. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2105–2109), and these negative results are predicted by the relatively weak binding shown by sedimentation partition chromatography. Because of the lability of receptor proteins, both the gel permeation and sucrose gradient assays are subject to serious artifacts: in particular, aggregated receptors can appear to be tightly DNA-bound. Short term co-sedimentation of receptors with the same DNA preparation sheared to two different sizes is shown to be necessary to distinguish between receptor aggregation and DNA binding. The low affinity, nonspecific, interaction of the estradiol receptor protein with DNA in vitro agrees with several in vivo observations; in particular, it might have been predicted from the careful studies by Williams and Gorski of the partitioning of the receptor between cell nucleus and cytoplasm (Williams, D., and Gorski, J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 3464–3468). Thus, although there are good reasons to believe that the receptor activates genes by interacting preferentially with a small number of specific sites on the genome, we believe that these interactions are obscured, both in vitro and in vivo, by a large background of nonspecific DNA binding.