Identification of a cyclin subunit required for the function of Drosophila P-TEFb.

P-TEFb is required for the transition from abortive elongation into productive elongation and is capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. We cloned a cDNA encoding the large subunit of Drosophila P-TEFb and found the predicted protein contained a cyclin motif. We now name the large subunit cyclin T and the previously cloned small subunit (Zhu, Y. R., Peery, T., Peng, J. M., Ramanathan, Y., Marshall, N., Marshall, T., Amendt, B., Mathews, M. B., and Price, D. H. (1997) Genes Dev. 11, 2622-2632) cyclin-dependent kinase 9 (CDK9). Recombinant P-TEFb produced in baculovirus-transfected Sf9 cells exhibited 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-sensitive kinase activity similar to native P-TEFb. Kc cell nuclear extract depleted of P-TEFb failed to generate long DRB-sensitive transcripts, but this activity was restored upon addition of either native or recombinant P-TEFb. Like other CDKs, CDK9 is essentially inactive in the absence of its cyclin partner. P-TEFb containing a CDK9 mutation that knocked out the kinase activity did not function in transcription. Deletion of the carboxyl-terminal domain of cyclin T in P-TEFb reduced both the kinase and transcription activity to about 10%. The CDK-activating kinase in TFIIH was unable to activate the CTD kinase activity of P-TEFb.