The Mediator complex associates with RNA polymerase II (RNAPII) at least partly via the RNAPII C-terminal repeat domain (CTD). This association greatly stimulates the CTD kinase activity of general transcription factor TFIIH, and subsequent CTD phosphorylation is involved in triggering promoter clearance. Here, highly purified proteins and a protein dissociation assay were used to investigate whether the RNAPII⅐Mediator complex (holo-RNAPII) can be disrupted by CTD phosphorylation, thereby severing one of the bonds that stabilize promoter-associated initiation complexes. We report that CTD phosphorylation by the serine 5-specific TFIIH complex, or its kinase module TFIIK, is indeed sufficient to dissociate holo-RNAPII. Surprisingly, phosphorylation by the CTD serine 2-specific kinase CTDK1 also results in dissociation. Moreover, the Mediator-induced stimulation of CTD phosphorylation previously reported for TFIIH is also observed with CTDK1 kinase. An unrelated CTD-binding protein, Rsp5, is capable of stimulating this CTD kinase activity as well. These data shed new light on mechanisms that drive the RNAPII transcription cycle and suggest a mechanism for the enhancement of CTD kinase activity by the Mediator complex. The C-terminal domain (CTD)2 of the largest RNA polymerase II subunit, Rpb1, is the target of dynamic phosphorylation/ dephosphorylation during the transcription cycle (1). A nonphosphorylated version of RNAPII enters promoters (2, 3), and the kinase activity associated with general transcription factor TFIIH somehow triggers promoter clearance by phosphorylation of serine 5 in the CTD repeat (Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 ) (4 -6). This is thought to be followed by phosphorylation of serine 2 by the CTDK1/pTEFb kinase and serine 5 dephosphorylation by Ssu72 phosphatase during transcript elongation (7-11). In all likelihood, Fcp1 phosphatase then removes the remaining CTD phosphorylation during or upon transcriptional termination, recycling the polymerase for a new round of transcription (12-15).The Mediator complex transduces signals from sequencespecific transcriptional regulators to the general transcription machinery (16 -18). The association of Mediator with RNAPII, and its function in transcription, depends on the RNAPII CTD (16). Mediator is able to bind an unphosphorylated glutathione S-transferase-CTD fusion protein in vitro (16) and can be displaced from RNAPII using the monoclonal antibody 8WG16, which specifically recognizes the CTD repeat (17,19). Apart from the CTD-interacting surfaces, Mediator also interacts with RNAPII domains outside of the CTD (20, 21).The observation that RNAPII enters the initiation complex in the unphosphorylated (RNAPIIA) form and leaves it in the hyperphosphorylated (RNAPIIO) form gave rise to the idea that the initiation to elongation transition is somehow facilitated by hyperphosphorylation of the CTD (3). The association with Mediator might be one of the mechanisms by which RNA-PIIA is tied to promoters. In crude yeast extracts, Mediator ...