Accurate in vitro initiation of beta-globin gene transcription in induced Friend-cell nuclei.
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.
Winicov
I
I
Weidner
D A
DA
Carlson
D P
DP
Ross
J
J
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D005622:Friend murine leukemia virus; D005914:Globins / Q000096:biosynthesis* / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D012315:RNA Caps; D012319:RNA Polymerase II / Q000378:metabolism; D012343:RNA, Transfer / Q000096:biosynthesis; D013873:Thionucleotides / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000546:Amanitins; D012315:RNA Caps; D013873:Thionucleotides; D005914:Globins; D012343:RNA, Transfer; D012319:RNA Polymerase II