ABSTRACT
Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.
Subject(s)
Fungal Proteins/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins , Acetyltransferases/chemistry , Acetyltransferases/genetics , Acetyltransferases/metabolism , Amino Acid Sequence , Base Sequence , DNA Primers/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Histone Acetyltransferases , Macromolecular Substances , Mediator Complex , Molecular Sequence Data , Multiprotein Complexes , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
RNA Polymerase II/chemistry , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/enzymology , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Chromatography, Affinity , Humans , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Phosphorylation , Protein Structure, Quaternary , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factor TFIIH , Transcription Factors/metabolismABSTRACT
Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.
