JBC, Vol. 251, Issue 16, 4843-4849, Aug, 1976

Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies on solubilized enzymes

J. C. Courvalin, M. M. Bouton, E. E. Baulieu, P. Nuret and P. Chambon

DNA-dependent RNA polymerases of immature and castrated rat uteri were studied after estradiol administration. The enzymes were solubilized from either whole uterus homogenate or nuclei and their activities were measured on an exogenous DNA template. alpha-Amanitin was used to distinguish alpha-amanitin-resistant from alpha-amanitin-senitive forms of the enzyme. The number of alpha-amanitin-sensitive RNA polymerase molecules was measured by a binding assay using labeled amanitin. In the first series of experiments RNA polymerases were solubilized from whole uterus homogenate. alpha-Amanitin-sensitive and -resistant activities were constant during the first 6 hours after estradiol treatment, followed by a late and moderate increase in their activities (50% at 12 hours for the resistant form and 40% at 24 hours for the sensitive form). The number of sensitive polymerase molecules evolved in an identical manner to its activity (+40% at 24 hours), suggesting that the increase in activity is due to the synthesis of new enzyme molecules. For both forms, no diffusible stimulatory factor was detected in the uterus of hormone-treated animals. In the second series of experiments, disrupted nuclei were washed with 0.15 M (NH4)2SO4 in order to release only enzyme molecules which were not firmly bound to DNA in a transcription complex. The amount of the sensitive form of polymerase which remains firmly bound to chromatin, was constant for 6 hours after estradiol administration and was doubled by 24 hours. The firmly bound alpha-amanitin-resistant activity was solubilized and was measured in the presence of an exogenous template. There was a progressive increase in activity first detectable in 1 to 2 hours, amounting to 50% at 6 hours and 100% at 24 hours. The reported results show that during the first 6 hours of hormone treatment: (a) the total content of RNA polymerases remains unchanged in the uterus; (b) the number of alpha-amanitin resistant molecules tightly bound to DNA increases progressively while the alpha-amanitin sensitive remains constant. At a later time (24 hours), an increase is observed both for the total amount of enzymes and for their fraction engaged in a transcription complex.
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