Abstract
Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.
Highlights
That was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB [3]
Additional evidence showing that PTEFb kinase functions as a positive elongation factor in vivo comes from studies with the HIV Tat protein that have shown that the catalytic activity of positive transcription elongation factor b (P-TEFb) is required for Tat-dependent stimulation of transcription elongation [9, 10]
It was previously reported that cyclin K interacts with RNA polymerase II and that immunoprecipitates obtained with cyclin K antibodies contained carboxyl-terminal domain (CTD) and Cdk kinase kinase activity [14]
Summary
That was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB [3]. P-TEFb is a DRB-sensitive kinase that is believed to stimulate the elongation potential of RNAPII by phosphorylating the CTD of RNAPII molecules that are engaged in early transcription elongation [5, 6]. It was recently suggested that P-TEFb-mediated phosphorylation of the CTD prevents the association of DSIF with RNAPII and thereby overcomes DSIFdependent repression [7]. The observation that the ability of several drugs to block CTD phosphorylation in vivo correlates with the ability of these compounds to inhibit P-TEFb in vitro strongly suggests that P-TEFb might function as a CTD kinase in vivo [8]. Additional evidence showing that PTEFb kinase functions as a positive elongation factor in vivo comes from studies with the HIV Tat protein that have shown that the catalytic activity of P-TEFb is required for Tat-dependent stimulation of transcription elongation [9, 10]. In this report we show that cyclin K associates with CDK9 in vivo and in vitro and demonstrate that cyclin K is a CDK9 regulatory subunit
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