The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

Abstract

Purified form A RNA polymerase [rat liver] and the endogenous, nuclear form A RNA polymerase incorporate 5-mercuri-uridine 5''-triphosphate (Hg-UTP) into RNA in vitro. The Km for both Hg-UTP and UTP are in the region of 10 .mu.M for the purified enzyme. The RNA products formed in nucleoli by endogenous RNA polymerase A have similar base compositions (G + C-rich) whether UTP or Hg-UTP is provided as the substrate in vitro. Sulfhydryl-Sepharose chromatography of RNA synthesized in vitro by nucleoli allows separation of this material from the endogenous RNA, when the former is synthesized in the presence of Hg-UTP. In-vitro-synthesized nucleolar RNA hybridizes with c0t renaturation time profiles similar to 28-S ribosomal RNA, when made with either Hg-UTP or UTP. Hybridization studies using DNA excess suggest that little competition occurs between the in vitro transcripts and the endogenous nucleolar RNA. Size analysis of in vitro transcripts show that although some degradation occurs during isolation, purification and hybridization of the RNA species, most of the RNA remains larger than 5 S throughout.