Abstract
Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5'-capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5'-capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / genetics
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Binding Sites
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Chromatin Immunoprecipitation
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Cyclin-Dependent Kinases / antagonists & inhibitors
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism*
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Enzyme Inhibitors / pharmacology
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Exoribonucleases / genetics
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Gene Expression Profiling
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Gene Expression Regulation, Fungal / drug effects
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Genome, Fungal*
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Glucose Transport Proteins, Facilitative / genetics
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Mutation
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Nucleotidyltransferases / genetics
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Nucleotidyltransferases / metabolism
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Oligonucleotide Array Sequence Analysis
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Phosphorylation
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Promoter Regions, Genetic / genetics
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Protein Binding
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RNA Polymerase II / genetics
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RNA Polymerase II / metabolism*
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Transcription, Genetic*
Substances
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Act1 protein, S cerevisiae
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Actins
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Enzyme Inhibitors
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Glucose Transport Proteins, Facilitative
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HXT1 protein, S cerevisiae
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RNA, Messenger
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Saccharomyces cerevisiae Proteins
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Cyclin-Dependent Kinases
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Kin28 protein kinase, S cerevisiae
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Nucleotidyltransferases
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RNA Polymerase II
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mRNA guanylyltransferase
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Exoribonucleases
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XRN1 protein, S cerevisiae