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Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis
J J Long1, A Leresche, R W Kriwacki
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.
Insights
Eukaryotic cells shut down nuclear transcription during mitosis. This repression, mediated by cdc2/cyclin B kinase, involves phosphorylation of key transcription factors like TFIIH, impacting RNA polymerase II activity.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Eukaryotic nuclear transcription is globally repressed during mitosis.
- This repression affects various cellular and viral gene promoters transcribed by RNA polymerase II.
Purpose of the Study:
- To investigate the molecular mechanisms underlying mitotic repression of transcription.
- To identify the specific components of the transcription machinery targeted by mitotic kinases.
Main Methods:
- In vitro transcription assays using cell extracts and purified components.
- Reconstituted transcription reactions with basal transcription factors and RNA polymerase II.
- Inhibition and rescue experiments using kinase inhibitors and specific substrates.
- Protein labeling and phosphorylation assays.
Main Results:
- Mitotic repression of transcription can be reproduced in vitro using cdc2/cyclin B kinase.
- Repression is reversed by p21Waf1/Cip1/Sdi1, confirming a role for protein phosphorylation.
- Multiple transcription factors, including TFIID and TFIIH, are inactivated by cdc2/cyclin B.
- TFIIH subunits (p62, p36) are direct phosphorylation targets; cdk7/cyclin H kinase activity is inhibited.
Conclusions:
- Mitotic transcription repression involves multiple phosphorylation targets within the transcription machinery.
- TFIIH is a major target for mitotic inactivation, impacting RNA polymerase II activity.
- The findings elucidate a key regulatory mechanism for gene expression control during the cell cycle.