Quantitation of elongating form A and B RNA polymerases in chick oviduct nuclei and effects of estradiol

Abstract

The number of elongating form A and B RNA polymerases in chick oviduct nuclei was estimated by measuring incorportaion of 3H-UTP into 3' termini of nascent RNA chains in the presence of heparin to inhibit initiation, and quantitation labeled uridine released from these termini after alkaline hydrolysis. The method corrects for conversion of UMP to uridine (U) during manipulations and for production of 3' termini by ribonucleases and phosphatases. The results indicate that a large fraction of RNA polymerases elongating in vivo is retained in isolated nuclei: per diploid genome, approximately 1 x104 form B and 2 x103 form A enzymes are present. These levels are sufficient to maintain normal in vivo rates of mRNA and rRNA synthesis, but the average density of packing of polymerases on DNA is considerably less than the maximum density predicted by Miller and Bakken (1972), suggesting that initiation of polymerases of DNA is a limiting factor in the control of transcription. Rates of elongation of polymerases in vitro are severely impaired, indicative of a loss of elongation factors during nuclear isolation. After 6 hr of estradiol treatment in vivo, the level of form A enzymes in ovoduct nuclei increases to 5 x 103; little change in the number of form B enzymes is seen.