Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe.
We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.
Spåhr
H
H
Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, 405 30 Göteborg, Sweden.
Bève
J
J
Larsson
T
T
Bergström
J
J
Karlsson
K A
KA
Gustafsson
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D002846:Chromatography, Affinity; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII
D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II