Activation of the Cyclin-dependent Kinase CTDK-I Requires the Heterodimerization of Two Unstable Subunits

Abstract

RNA polymerase II CTD kinases are key elements in the control of mRNA synthesis. They constitute a family of cyclin-dependent kinases activated by C-type cyclins. Unlike most cyclin-dependent kinase complexes, which are composed of a catalytic and a regulatory subunit, the yeast CTD kinase I complex contains three specific subunits: a kinase subunit (Ctk1), a cyclin subunit (Ctk2), and a third subunit (Ctk3) of unknown function that does not exhibit any similarity to known proteins. Like the Ctk2 cyclin that is regulated at the level of protein turnover, Ctk3 is an unstable protein processed through a ubiquitin-proteasome pathway. Interestingly, Ctk2 and Ctk3 physical interaction is required to protect both subunits from degradation, pointing to a new mechanism for cyclin turnover regulation. We also show that Ctk2 and Ctk3 can each interact independently with the kinase. However, despite the formation of CDK/ cyclin complexes in vitro, the Ctk2 cyclin is unable to activate its CDK: both Ctk2 and Ctk3 are required for Ctk1 CTD kinase activation. The different specific features governing CTDK-I regulation probably reflect requirement for the transcriptional response to multiple growth conditions. The C-terminal domain (CTD) 1 of the large subunit of RNA polymerase II contains a repeated heptapeptide that is highly phosphorylated in a portion of the molecules in the cell. This domain plays an essential role in the control of mRNA synthesis, as well as RNA processing, and its phosphorylation is a key feature of its function (1). The formation of initiation complexes on promoter DNA involves RNA polymerase II molecules with unphosphorylated CTD, whereas the CTD becomes phosphorylated during or after the transition to elongation. Three CTD kinases are involved in the regulation of RNA polymerase II transcription in the yeast Saccharomyces cerevisiae, all being cyclin-dependent kinases (CDKs).The first yeast CTD kinase, Kin28 (2), is associated with cyclin Ccl1 (3) and is required for transcription of most genes (4). This complex, like its human counterpart the Cdk7 kinase complex (5), is a component of the general transcription factor TFIIH (6, 7). Kin28 is an essential kinase that has been shown to phosphorylate the CTD only after the RNA polymerase II is associated with promoter DNA, thus promoting the elongation mode (8). Kin28 is also required for proper capping enzyme targeting in vivo (9). The second yeast CDK implicated in CTD phosphorylation, the nonessential Srb10/Ume5/Ssn3 kinase and its cyclin partner Srb11/Ume3/Ssn8, is a component of the holoenzyme (10). The Srb10/11 complex is structurally related to mammalian Cdk8 kinase and its associated cyclin C (11). Several studies established that the Srb10 kinase is implicated in the transcriptional repression of specific sets of genes (4, 12-15). Hengartner et al. (8) showed that Srb10 is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. Srb10 has also been i...

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