Histone Acetyltransferase and Protein Kinase Activities Copurify with a Putative Xenopus RNA Polymerase I Holoenzyme Self-Sufficient for Promoter-Dependent Transcription

Abstract

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of ∼2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.

ACKNOWLEDGMENTS

We thank Liang Annie Shen for excellent technical assistance, Jerry Workman (Pennsylvania State University) for the gift of purified HeLa histones, Neil Osheroff (Vanderbilt University) for the gift of antibodies against Drosophila CKII, and Paul Labhart (Scripps Research Institute) and Brian McStay (University of Dundee) for gifts of antibodies against Xenopus TBP. We are grateful to Larry Rothblum (Geisinger Clinic) for helpful discussions and for sharing unpublished results and materials.

This work was supported by NIH grant 5-RO1-GM50910 to C.S.P. Annie-Claude Albert was supported, in part, by a W. M. Keck Fellowship awarded through the Washington University School of Medicine.