Nuclear transcription in vitro. Sensitivity to inhibition by ribosyldichlorobenzimidazole and rifamycin AF/013

Abstract

L cell nuclear preparations were shown to transcribe RNA for periods up to 1 h at 37 degrees C. Nearly 70% of the transcription products were sensitive to inhibition by 1 microgram/mL of alpha-amanitin, indicating that they were transcribed by RNA polymerase II. Analysis of polyphosphorylated termini of in vitro synthesized RNA showed the presence of a phosphatase activity which prevents quantitative recovery of these termini. The finding of in vitro labeled polyphosphorylated termini in RNA greater than 12 S after short periods of incubation shows initiation in vitro for this size class. The labeling of these polyphosphorylated termini is decreased in the presence of rifamycin AF/013. The use of two apparent inhibitors of initiation, rifamycin AF/013 and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), has allowed detection of in vitro initiated transcripts of heterogeneous nuclear RNA. Both of these inhibitors act primarily at later times of incubation, in contrast to alpha-amanitin which acts on elongation and inhibits in vitro RNA synthesis immediately. The selective pattern of DRB inhibition on hnRNA is retained in vitro and some accumulation of large-size molecules is observed. It can be estimated that about 30% of the greater than 12S hnRNA sequences transcribed in vitro are sensitive to DRB and 48% of greater than 12S RNA are sensitive to rifamycin AF/013 inhibition.