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The structure and substrate specificity of human Cdk12/Cyclin K

Christian A Bösken¹, Lucas Farnung², Corinna Hintermair³

¹1] 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.

Nature Communications

|March 26, 2014

Summary

Cyclin-dependent kinase 12 (Cdk12) interacts with its cyclin partner CykK to regulate RNA polymerase II transcription. This study reveals the Cdk12/CycK structure and its specific CTD phosphorylation activity.

Area of Science:

Biochemistry
Molecular Biology
Structural Biology

Background:

Phosphorylation of RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases (CDKs) is crucial for transcription.
Understanding the structural basis of CDK-CTD interactions is key to deciphering transcriptional regulation.

Purpose of the Study:

To determine the crystal structure of Cdk12/CycK.
To analyze the substrate recognition requirements of Cdk12/CycK.
To investigate the regulation and activity of Cdk12/CycK in transcription.

Main Methods:

X-ray crystallography to determine the Cdk12/CycK structure.
Biochemical assays to assess CTD substrate phosphorylation.
Enzyme kinetics to compare Cdk12/CycK and Cdk9/CycT activity.
Inhibition assays using Flavopiridol.

Main Results:

The crystal structure revealed an open conformation for active Cdk12/CycK, distinct from cell cycle kinases.
A conserved C-terminal extension in Cdk12 interacts with the ATP ribose via an HE motif and polybasic cluster.
Cdk12/CycK preferentially phosphorylated CTD substrates prephosphorylated at Ser7.
Flavopiridol showed higher potency against Cdk12 than Cdk9, and T-loop phosphorylation required a Cdk-activating kinase.

Conclusions:

The findings elucidate the structural basis for Cdk12/CycK activity and substrate specificity in CTD phosphorylation.
Results suggest a regulatory mechanism for RNA polymerase II elongation involving a cascade of CTD kinases.

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