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.