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

A systematic approach to the in vitro measurement of HNA-dependent RNA polymerase activities in nuclei from immature rat uterus has been undertaken in order to evaluate the early effects of estradiol. The experimental conditions include excess of nucleoside .i’-triphosphate and short time assays (6 min) at low temperature (25”) in order to obtain the maximal velocity of the reaction and to minimize RNase activity. Nucleotide incorporation has been measured in the absence (low ionic strength medium) or in the presence (high ionic strength medium) of 0.25 ~1 ammonium sulfate under various divalent cation concentrations, either in the presence or absence of cu-amanitin. The cr-amanitin-resistant RNA polymerase activity of nuclei is identical under three conditions, low ionic strength with either Mg” or Mr?+ (4 mM) and high ionic strength in the presence of 4 ma Mns+. cY-Amanitin-sensitive activity measured at high ionic strength is about 10 times greater than the activity measured at low ionic strength in the presence of 4 rnvr Mn”‘. Under all experimental conditions used in these studies, in vitro initiation of new RNA chains is negligible. Estradiol administration leads to an early (1 to 2 h) increase (50%;) in cu-amanitin-resistant activity under all experimental conditions. The a-amanitin-sensitive activity exhibits a similar evolution (+lOO% at 2 h) when measured under low ionic strength conditions. However, the same activity, measured under high ionic strength conditions, is constant during the first 6 h after hormone treatment. 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 the number of cu-amanitinresistant molecules engaged in the process of transcription, while the number of cY-amanitin-sensitive molecules remains constant. The increase in Lu-amanitin-sensitive activity obtained under low ionic strength conditions can be interpreted either by an increase in template activity, or by