Primer-dependent eukaryotic RNA polymerase capable of accurate transcription from the adenovirus major late promoter in a reconstituted system

Abstract

A sensitive assay for detection of eukaryotic RNA polymerase II has been developed. This assay depends on the ability of polymerase II to elongate a small RNA primer, oligo(U), hybridized to a single-stranded homopolymeric DNA template, poly(dA). The poly(dA).oligo(U)-dependent RNA polymerase II from calf thymus has been purified approximately 10,000-fold using this assay. The purified enzyme contains four polypeptides of apparent Mr 180,000, 140,000, 24,000, and 16,000 and is fully active in accurate initiation of transcription from the adenovirus major late promoter in the presence of transcription factors from HeLa cells. The poly(dA).oligo(U)-dependent RNA polymerase activity can be detected in crude cell extracts from a variety of tissue culture cells and appears to be largely due to polymerase II, since 90-95% of this activity is inhibited by alpha-amanitin at a concentration of 1 microgram/ml.