In vitro transcription in plant nuclei

Abstract

Isolated plant nuclei can be used for fundamental studies on the transcription apparatus. Total RNA polymerase activity can be measured using plant nuclei, and by using different α-amanitin concentrations in the enzyme assay, the individual RNA polymerase I, II, and in activities can be measured (1-5). The assay procedure involves the incubation of nuclei in the presence of the four substrates for RNA synthesis: ATP, GTP, CTP, and UTP. If one of these precursors is supplied as a radio-labeled molecule, transcription can be detected as incorporation of radioactivity into acid-insoluble material. Following incubation, transcription products are precipitated with TCA, collected and washed on glass fiber filter discs, and counted by liquid scintillation counting.