Selective kinase inhibition shows that Bur1 (Cdk9) phosphorylates the Rpb1 linker in vivo

Abstract

Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdkl2/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at Serines 5 and 7, which are known target sites, but also at Serine 2. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for Serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tSH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for this modification in vivo.