Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription.
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.
Albert
A C
AC
Biology Department, Washington University, St. Louis, Missouri 63130, USA.
Denton
M
M
Kermekchiev
M
M
Pikaard
C S
CS
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
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