REPRESSION OF TFIIH TRANSCRIPTIONAL ACTIVITY AND TFIIH-ASSOCIATED CDK7 KINASE-ACTIVITY AT MITOSIS

Nuclear transcription is repressed when eukaryotic cells enter mitosis . Mitotic repression of transcription of various cellular and viral ge ne promoters by RNA polymerase II can be reproduced in vitro either wi th extracts prepared from cells arrested at mitosis with the microtubu le polymerization inhibitor nocodazole or with nuclear extracts prepar ed from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcr iption in reconstituted transcription reactions with biochemically pur ified and recombinant basal transcription factors and RNA polymerase I I. The cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) can rever se the effect of cdc2/cyclin B kinase, indicating that repression of t ranscription is due to protein phosphorylation. Transcription rescue a nd inhibition experiments with each of the basal factors and the polym erase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase, For an activated promoter, ta rgets of repression are TFIID and TFIIH, while for a basal promoter, T FIIH is the major target for mitotic inactivation of transcription. Pr otein labeling experiments indicate that the p62 and p36 subunits of T FIIH are in vitro substrates for mitotic phosphorylation. Using the ca rboxy-terminal domain of the large subunit of RNA polymerase II as a t est substrate for phosphorylation, the TFIIH-associated kinase, cdk7/c yclin H, is inhibited concomitant,vith inhibition of transcription act ivity. Our results suggest that there exist multiple phosphorylation t argets for the global shutdown of transcription at mitosis.