Accurate in vitro initiation of beta-globin gene transcription in induced Friend-cell nuclei

Abstract

The initiation of transcription by RNA polymerase II in isolated murine erythroleukemia cell nuclei was investigated by isolating newly synthesized gamma-thio (gamma-S-)-triphosphate-labeled transcripts by Hg-agarose chromatography. The 5' terminus of transcripts initiated in vitro with [gamma-35S]ATP or [gamma-35S]GTP was identified as the thiotetraphosphate in alkaline hydrolysis products from Hg-agarose-selected RNA. Additional control experiments analyzing the nuclear transcription of two well characterized tRNA genes showed that each gene was initiated with the proper triphosphate, either gamma-S-ATP or gamma-S-GTP, indicating little, if any, exchange of the gamma-S-labeled substrate to the other triphosphates. As determined by S1 mapping, newly synthesized beta-globin gene transcripts initiate only with gamma-S-ATP. Their 5'-terminus is located at the cap site, and their synthesis is inhibited by 1 microgram alpha-amanitin/ml. In reactions containing gamma-S-ATP but not gamma-S-GTP, several additional initiation sites are observed that are located in the 5'-flanking region. We conclude that RNA polymerase II can initiate transcription at the cap site in isolated nuclei.