Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II.
Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. Here, we demonstrate that cyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, we found that only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, we found that the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, our data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo.
Napolitano
G
G
Department of Genetics, Molecular and General Biology, University of Naples 'Federico II' and International Institute of Genetics and Biophysics, CNR, Via Mezzocannone 8, 80134, Naples, Italy.
Majello
B
B
Licciardo
P
P
Giordano
A
A
Lania
L
L
D001665:Binding Sites; D002460:Cell Line; D056906:Cyclin T; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism / Q000502:physiology; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C113028:CCNK protein, human; C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D017346:Protein Serine-Threonine Kinases; C095686:CDK8 protein, human; C467969:CDK9 protein, human; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human