Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.
The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden. endre.egyhazi@cmb.ki.se
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Egyházi
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Ossoinak
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Lee
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Greenleaf
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Mäkelä
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Pigon
D000818:Animals; D002683:Chironomidae / Q000201:enzymology / Q000235:genetics*; D002875:Chromosomes / Q000201:enzymology; D018844:Cyclin-Dependent Kinases; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D017344:Genes, Insect; D018869:Heat-Shock Response / Q000235:genetics*; D019476:Insect Proteins / Q000235:genetics; D007814:Larva; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D012319:RNA Polymerase II / Q000032:analysis / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012469:Salivary Glands / Q000166:cytology / Q000201:enzymology; D014158:Transcription, Genetic / Q000502:physiology*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D019476:Insect Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D004004:Dichlororibofuranosylbenzimidazole; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase