SRP311463 PRJNA716055 GSE169271 Localized phosphorylation of RNA Polymerase II by G1 cyclin-Cdk promotes cell cycle entry The cell cycle is thought to be initiated by cyclin-dependent kinases (Cdk) inactivating transcriptional inhibitors of cell cycle gene-expression. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate Whi5, thereby releasing the transcription factor SBF and committing cells to division. Here, we report that Cln3-Cdk1 does not phosphorylate Whi5, but instead phosphorylates the RNA Polymerase II subunit Rpb1's C-terminal domain (CTD) on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters(8) and Cln3's function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to promote transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these processes. Overall design: ChIP-seq GSE169271