Utilization of 5-alkyl UTPs by DNA-dependent RNA polymerase I and II purified from cherry salmon (Onchorhynchus masou) liver.

DNA dependent RNA polymerase II was purified to approximately 8300 fold from sonicated nuclear extract of cherry salmon (Onchorhynchus masou) liver by the following purification steps: polyethylene glycol treatment, DEAE=Sephadex A-25 column chromatography, heparin-Sepharose column chromatography, and affinity chromatography on DNA-cellulose. Final preparation of this enzyme has a specific activity of 157 nmole UMP incorporation into RNA per mg of protein per 10 min at 25 degrees. RNA polymerase I was also purified to approx. 3800 fold in a similar manner. Its specific activity was calculated as 26.2 nmole/mg/10 min. Utilization of various UTPs of these enzymes was studied by substitution experiments under the condition of limited synthesis. 5-Methyl UTP (rTTP) could be utilized by the RNA polymerase I 1.7 fold more efficiently compared with UTP. In contrast, the RNA polymerase II recognized rTTP as a substrate as efficiently as UTP. Similar experiments using other alkyl UTPs have been performed.