Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription.
Cdc2 kinase triggers the entry of mammalian cells into mitosis, the only cell cycle phase in which transcription is globally repressed. We show here that Cdc2 kinase phosphorylates components of the RNA polymerase II transcription machinery including the RNA polymerase II carboxy-terminal repeat domain (CTD). To test specifically the effect of CTD phosphorylation by Cdc2 kinase, we used a yeast in vitro transcription extract that is dependent on exogenous RNA polymerase II that contains a CTD. Phosphorylation was carried out using immobilized Cdc2 so that the kinase could be removed from the phosphorylated polymerase. ATP gamma S and Cdc2 kinase were used to produce an RNA polymerase IIO that was not detectably dephosphorylated in the transcription extract. RNA polymerase IIO produced in this way was defective in promoter-dependent transcription, suggesting that phosphorylation of the CTD by Cdc2 kinase can mediate transcription repression during mitosis. In addition, we show that phosphorylation of pol II with the human TFIIH-associated kinase Cdk7 also decreases transcription activity despite a different pattern of CTD phosphorylation by this kinase. These results extend previous findings that RNA polymerase IIO is defective in preinitiation complex formation. Here we demonstrate that phosphorylation of the CTD by cyclin-dependent kinases with different phosphoryl acceptor specificities can inhibit transcription in a CTD-dependent transcription system.
Gebara
M M
MM
Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.
Sayre
M H
MH
Corden
J L
JL
D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000737:chemistry* / Q000302:isolation & purification*; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics*
D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II