Abstract
Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Binding Sites
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Gene Expression Regulation, Fungal
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Holoenzymes / chemistry
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Holoenzymes / genetics
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Holoenzymes / metabolism
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Magnetic Resonance Spectroscopy
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Methyl Methanesulfonate / pharmacology
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Mutation
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Phenotype
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Phosphoprotein Phosphatases / genetics
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Phosphoprotein Phosphatases / metabolism*
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Phosphorylation
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Protein Binding
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Protein Structure, Tertiary / genetics
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RNA Polymerase II / chemistry
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism
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RNA, Messenger / biosynthesis
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Recombinant Fusion Proteins / metabolism
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Repetitive Sequences, Nucleic Acid
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Transcription Factor TFIIB
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Transcription Factors / chemistry*
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcription Factors, TFII*
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Transcriptional Activation
Substances
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Holoenzymes
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RNA, Messenger
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Recombinant Fusion Proteins
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Transcription Factor TFIIB
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Transcription Factors
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Transcription Factors, TFII
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Methyl Methanesulfonate
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RNA Polymerase II
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Phosphoprotein Phosphatases
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carboxy-terminal domain phosphatase
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transcription factor TFIIF