Promoter-proximal pausing by RNA polymerase II (Pol II) ensures both gene-specific regulation and RNA quality control. Structural considerations suggested initiation factor eviction would be required for elongation factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7—part of TFIIH—increases TFIIE retention, prevents DRB-sensitivity inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9—cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events—Pol II carboxyl-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation—in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3′-end formation. Cdk7 thus acts through TFIIE and DSIF to establish and through P-TEFb to relieve barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and co-transcriptional RNA maturation.

Cell division is controlled by cyclin-dependent kinases (CDKs). In metazoans, S phase onset coincides with activation of Cdk2, whereas Cdk1 triggers mitosis. Both Cdk1 and -2 require cyclin binding and T loop phosphorylation for full activity. The only known CDK-activating kinase (CAK) in metazoans is Cdk7, which is also part of the transcription machinery. To test the requirements for Cdk7 in vivo, we replaced wild-type Cdk7 with a version sensitive to bulky ATP analogs in human cancer cells. Selective inhibition of Cdk7 in G1 prevents activation (but not formation) of Cdk2/cyclin complexes and delays S phase. Inhibiting Cdk7 in G2 blocks entry to mitosis and disrupts Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation-cyclin binding and T loop phosphorylation-are mutually dependent. Therefore, by combining chemical genetics and homologous gene replacement in somatic cells, we reveal different modes of CDK activation by Cdk7 at two distinct execution points in the cell cycle.

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5 ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5 pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3 ends, it was detected within genes and 3-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.Dynamic modification of the RNA polymerase II (Pol II) C-terminal domain (CTD) by phosphorylation and dephosphorylation plays important roles in controlling both transcription and cotranscriptional RNA processing (9, 31). There are 52 heptad repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 in the human CTD that can be phosphorylated cotranscriptionally on serines 2, 5, and 7 (S2, S5, and S7, respectively) (6, 31). S5 phosphorylation normally occurs early in the transcription cycle coincident with initiation, whereas S2 phosphorylation predominates later, during elongation and termination (13,20). The complex pattern of heptad repeat phosphorylation serves in part to control binding of partner proteins, including elongation factors, RNA processing factors (31), and chromatin modifiers (25, 39). Ser5 phosphorylation enhances cotranscriptional mRNA capping (17), and Ser2 facilitates 3Ј-end formation by cleavage/polyadenylation (1, 4, 29). Ser7 phosphorylation has been specifically implicated in formation of U snRNA 3Ј ends but is also found on mRNA coding genes (10) (6). Exactly how S2, S5, and S7 phosphorylation affect initiation, elongation, and termination remains poorly understood.It is generally thought that the positive transcription elongation factor PTEFb (Cdk9/cyclin T [CycT]) is the principal S2 kinase and that Cdk7 associated with TFIIH is the principal S5 kinase (31,32,38). How Cdk7 affects CTD phosphorylation on metazoan genes in vivo is still an unresolved question, and the S7 kinase is yet to be identified. In the only previous investigation of Cdk7 effects on CTD phosphorylation at the site of transcription in a multicellular organism, phospho-S5 and t...

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1 wt ) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1 as ). When cultured in the presence of these analogs, Plk1 as cells accumulate in prometaphase with defects that parallel those found in PLK1 ⌬/⌬ cells. In addition, acute treatment of Plk1 as cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.cell division ͉ Ect2 ͉ knockout ͉ mitosis

Cdk7 has been shown previously to be able to phosphorylate and activate many different Cdks in vitro. However, conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive. Adding to the controversy is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7. Furthermore Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but as the catalytic subunit of TFIIH. Vertebrate Cdk7 is also known to be part of TFIIH. Therefore, in the absence of better genetic evidence, it was proposed that the CAK activity of Cdk7 may be an in vitro artifact. In an attempt to resolve this issue, we cloned the Drosophila cdk7 homolog and created null and temperature-sensitive mutations. Here we demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism. We show that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks.