Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.