PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation

The transcription cycle is regulated by dynamic changes in RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphorylation, which are crucial for gene expression. However, the mechanisms regulating the transcription-specific cyclin-dependent kinases (CDKs) during the transcription cycle remain poorly understood. Here, we show that human CDK12 co-phosphorylates CTD Serine₂ and Serine₅. This di-phosphorylated Serine₂-Serine₅ CTD mark may then act as a precursor for Serine₂ mono-phosphorylated CTD through Serine₅ de-phosphorylation. Notably, CDK12 is specifically regulated by association with the elongation-specific factor PAF1 complex (PAF1C), in which the CDC73 subunit contains a metazoan-specific peptide motif, capable of allosteric CDK12/cyclin K activation. This motif is essential for cell proliferation and required for normal levels of CTD phosphorylation in chromatin, and for transcript elongation, particularly across long human genes. Together, these findings provide insight into the mechanisms governing RNAPII phospho-CTD dynamics that ensure progression through the human transcription cycle.