DNase I digestion of chromatin from avian liver nuclei liberates DNA-dependent RNA polymerase II.

In an effort to develop mild conditions for the isolation of DNA-dependent RNA polymerase II, we have used DNase I covalently coupled to Sepharose 4B to digest chromatin from hypotonically lysed nuclei from rooster liver. The RNA polymerase II released was at least 2 times more active in in vitro transcription than was RNA polymerase II prepared by the conventional method of sonication of chromatin in buffers containing high salt. The numbers of RNA polymerase II molecules in RNA polymerase preparations prepared both by DNase I-Sepharose digestion and by sonication were determined by titration with [3H]amanitin and were similar in both preparations, indicating that the two methods were equally efficient at liberating RNA polymerase from the chromatin but that treatment with DNase I-Sepharose yielded higher levels of enzymatic activity. In order to identify RNA polymerase II in complex mixtures of proteins, we have utilized the technique of binding r3H]amanitin to RNA polymerase II followed by electrophoresis on gradient polyacrylamide gels under nondenaturing conditions. We have identified two forms of RNA polymerase II, having molecular weights of 640,000 and 550,000. The release of highly active eukaryotic RNA polymerases by the very gentle treatment of chromatin from lysed nuclei with DNase I-Sepharose may facilitate the reconstitution of an in vitro transcription system using RNA polymerase II.