Progesterone receptor binding to DNA: studies by sedimentation velocity methods.
Chicken oviduct progesterone receptor subunit A (Mr = 79000) was partially purified as a hormone-receptor complex labeled with [3H]progesterone. These complexes adsorb to DNA-cellulose; sucrose gradient ultracentrifugation showed a sedimentation coefficient of 3.6 S. Receptors were mixed with [32P]DNA fragments of various types derived from bacterial plasmid pBR322. DNA structures tested included linear double-stranded and single-stranded molecules as well as supercoiled intact plasmids. [3H]Receptors were mixed in various molar ratios with [32P]DNA (nominal sed. coeff. = 18 S) and the mixtures were analyzed by sedimentation velocity ultracentrifugation performed in 5-20% sucrose gradients. In the presence of excess DNA, all of the [3H]receptor sedimented with the DNA fraction. Titrations revealed an apparent Kdiss between 1 and 5 nM. Distribution of receptor counts over the slower-sedimenting edge of the DNA peaks allowed estimation of the half-life of these complexes, which was in excess of 35 min at 0 degree C. Double-label counting of [3H]receptor-[32P]DNA complexes revealed an increase in the sedimentation coefficient of linear double-stranded DNA as increasing receptor input was used. Analysis of this S-value shift showed evidence for cooperative receptor binding isotherms, causing dramatic compaction (or other topological constraints) on the DNA. A speculative schematic model of receptor A-DNA interaction is presented, consistent with the experimental results.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010057:Oviducts / Q000032:analysis; D011374:Progesterone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism*
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D011980:Receptors, Progesterone; D011374:Progesterone; D004247:DNA