title: 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 approximately 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.
authors.affiliation: Biology Department, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Albert
authors.forename: M
authors.initials: M
authors.lastname: Denton
authors.forename: M
authors.initials: M
authors.lastname: Kermekchiev
authors.forename: C S
authors.initials: CS
authors.lastname: Pikaard
mesh_terms: D000123:Acetyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D051548:Histone Acetyltransferases; D020035:Holoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D035463:Pol1 Transcription Initiation Complex Proteins; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012318:RNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000302:isolation & purification; D014158:Transcription, Genetic; D014982:Xenopus laevis
chemical_list: D004268:DNA-Binding Proteins; D020035:Holoenzymes; D035463:Pol1 Transcription Initiation Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C063844:transcription factor UBF; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; D011494:Protein Kinases; D012318:RNA Polymerase I