Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics

CDK7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) CDK7 able to use bulky ATP derivatives. Cdk7-AS-cyclin H-Mat1 phosphorylates approximately 10-15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown CDK7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated CDK7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of CDK7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, CDK7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.