Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies with whole nuclei.

Abstract

Immature female Sprague-Dawley rats (19-21 days old 50 gm) were used to systematically study DNA-dependent RNA polymerase activity in nuclei from the uterus after administration of 1 mcg estradiol dissolved in .1 ml of 9% NaC1 solution. Controls received only the saline solution. Experimental conditions included an excess of nucleoside 5-triphosphate and short-time assays (6 minutes) at low temperature to obtain the maximal velocity of the reaction and to minimize RNase activity. Nucleotide incorporation was measured in the absence (low ionic strength medium) or the presence (high ionic strength medium) of .25 M ammonium sulfate under various divalent cation concentrations either in the presence or absence of alpha-amanitin. The alpha-amanitin-resistant RNA polymerase activity of nuclei was identical when measured under low ionic strength conditions with either Mg or Mn and under high ionic strength in the presence of Mn (all at 4 mM concentrations). At high ionic strength alpha-amanitin-sensitive activity is about 10 times greater than activity measured at low ionic strength Mn. In all the experiments initiation of new RNA chains was negligible. Estradiol administration leads to: a 50% increase in alpha-amanitin-resistant activity within 1-2 hours under all experimental conditions; a 100% increase by 2 hours in alpha-amanitin-sensitive activity under low ionic strength conditions; but no change in alpha-amanitin-sensitive activity at 6 hours under high ionic strength conditions. Since enzyme activity measured under high ionic strength conditions mainly reflects the number of enzyme molecules engaged in transcription these results suggest that estradiol leads to an early increase in number of alpha-amanitin-resistant molecules engaged in the process of transcription while the number of alpha-amanitin-sensitive molecules remains constant. The increase in alpha-amanitin-sensitive activity obtained under low ionic strength conditions can be interpreted as either an increase in template activity or an activation of the molecules already engaged in the process of transcription or both.(AUTHORS MODIFIED)