Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

] Group Physical Biochemistry Center of Advanced European Studies and Research Ludwig Erhard Allee 2 Bonn 53175 Germany [2] Department of Physical Biochemistry Max Planck Institute of Molecular Physiology Otto Hahn Strasse 11 Dortmund 44227 Germany

Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

Department of Molecular Epigenetics Helmholtz Center Munich Center for Integrated Protein Science Marchioninistrasse 25 München 81377 Germany

Department of Physical Biochemistry Max Planck Institute of Molecular Physiology Otto Hahn Strasse 11 Dortmund 44227 Germany

Department of Structural and Chemical Biology Icahn School of Medicine at Mount Sinai New York New York 10029 USA

Group Physical Biochemistry Center of Advanced European Studies and Research Ludwig Erhard Allee 2 Bonn 53175 Germany

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