Abstract
The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes. In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant. In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Chromatography, Affinity
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Cyclins / biosynthesis
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / metabolism
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Fungal Proteins / genetics
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Fungal Proteins / isolation & purification
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Fungal Proteins / metabolism*
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Gene Deletion
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Macromolecular Substances
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Mass Spectrometry
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Molecular Weight
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Multienzyme Complexes / chemistry
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Multienzyme Complexes / metabolism
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Nuclear Proteins / chemistry*
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Nuclear Proteins / genetics
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Nuclear Proteins / isolation & purification
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Nuclear Proteins / metabolism
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Phenotype
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Proteasome Endopeptidase Complex
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Protein Binding
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RNA Polymerase II / chemistry*
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RNA Polymerase II / genetics
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RNA Polymerase II / isolation & purification
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RNA Polymerase II / metabolism
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Reproducibility of Results
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism*
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Suppression, Genetic
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TATA-Box Binding Protein*
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Transcription Factors*
Substances
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CLN1 protein, S cerevisiae
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Cyclins
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Fungal Proteins
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Macromolecular Substances
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Multienzyme Complexes
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Nuclear Proteins
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PAF1 protein, S cerevisiae
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RTF1 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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TATA-Box Binding Protein
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Transcription Factors
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RNA Polymerase II
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex