Abstract
Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/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, the known target sites. However, serine 2 phosphorylation was also abrogated, supporting an obligatory sequential phosphorylation mechanism. 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 tandem SH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for these modifications in vivo.
Highlights
Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription
The mutated genes were introduced into yeast using plasmid shuffling, and protein expression levels were tested using a triple hemagglutinin (HA3) tag introduced onto the C terminus
Immunoblotting showed that Kin28-irreversibly sensitized” (IS) and Ctk1-IS proteins were expressed at levels similar to their wild-type counterparts (Fig. 1B)
Summary
Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin, Cdk9/Bur, and Cdk12/Ctk phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. Cdk7/Kin assembles into the preinitiation complex (PIC) as part of the basal transcription factor TFIIH It phosphorylates the C-terminal domain (CTD) of polymerase subunit Rpb, primarily on serine 5 of the repeated consensus sequence YSPTSPS. This phosphorylation triggers several events near 5= ends of genes: it dissociates the Mediator complex that chaperones RNA polymerase II (RNApII) into the PIC, while creating binding sites for the mRNA capping enzyme, the Nrd1/Nab early termination complex, and the Set1/COMPASS histone methyltransferase. Cdk9/Bur may phosphorylate serine 2 of the Rpb CTD at a low level during early elongation [8], other experiments suggest that it targets Ser (reviewed in reference 6). We extend the IS approach to Bur1/Cdk and Ctk1/Cdk
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