Abstract
Transcription of a cloned rat rDNA containing the transcription initiation region was studied using purified fractions derived from whole cell extract. The cell extract obtained from rat mammary adenocarcinoma cells was fractionated successively on DEAE-Sephadex and heparin-Sepharose columns. The fractions eluted by 175 mM (NH4)2SO4 (DE-B) from the DEAE-Sephadex column and those eluting with RNA polymerase I on the subsequent heparin-Sepharose column (HS-B) were used in a run-off transcription assay using Xho I-cleaved cloned rat rDNA. Both fractions that contained greater than 90% of the RNA polymerase I activity produced the anticipated 635-nucleotide-long transcript. Fractionation of fraction DE-B on a phosphocellulose column also resulted in a RNA polymerase I-containing complex that, by itself, could transcribe the rDNA accurately. The major polypeptides obtained after NaDodSO4/PAGE of fraction HS-B had Mr values of 190,000, 120,000, 65,000, 42,000, and 25,000, corresponding to the major subunits of RNA polymerase I. Purified nuclear RNA polymerase I did not produce the correct transcript. These studies have shown that a purified fraction containing RNA polymerase I from whole cells can support accurate transcription of rDNA.