Mammalian RNA polymerases and their selective inhibition by amanitin

Abstract

Summary 1. RNA polymerase activities of rat liver nuclei have been separated by DEAE-Sephadex chromatography. In addition to the major three peaks of enzyme activities reported by other investigators in whole nuclear extracts, at least one more activity has been resolved by chromatography of the enzyme extract prepared by a comparatively mild procedure. A homogeneous assay system was employed throughout using liver nuclear DNA instead of calf thymus DNA as the template. In some experiments, additional enzyme activities were found after enzyme C was eluted. 2. α -Amanitin inhibits the enzyme activity solubilized from nucleoplasm whereas the enzyme extracted from isolated nucleoli remains unaffected even at a very high concentration of the toxin. 3. α -Amanitin has been shown to inhibit RNA synthesis by binding to the enzyme and it acts at a step after the initiation of the RNA synthesis. 4. Administration of α -amanitin in vivo to rats (30 μ g/100 g body weight), however, inhibits the incorporation of 14 C-orotic acid into both nucleolar and nucleoplasmic (extranucleolar) RNA synthesis within 1 hr. However, the inhibition of ribosomal RNA synthesis in the nucleolus is reversed after 3 hr whereas the synthesis of non-ribosomal RNA in the nucleoplasm remains inhibited at this time. Correspondingly, the 45S optical density peak disappears within 1 hr of treatment with the toxin and reappears dramatically to the control level within 3 hr. In agreement with the in vivo observations, RNA polymerase activities solubilized from the nucleolar and nucleoplasmic fractions were both inhibited within 1 hr. However, the recovery of the nucleolar polymerase activity is not as fast as that of the labeling of nucleolar RNA in vivo . 5. The enzyme activities of the control and α -amanitin-treated samples were also resolved by column chromatography of the nuclear extracts. The enzyme activity in the void volume of the eluent and the activity of enzyme B (nucleoplasmic polymerase) show extensive loss of activity after amanitin treatment, whereas enzyme A (nucleolar polymerase) shows some inhibition. The remaining peaks of activity were essentially unaffected. The complicating factors involved in in vivo experiments have been discussed. The experiments suggest that amanitin inhibits ribosomal RNA synthesis in vivo through some extranucleolar mechanism.