Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences

Area of Science:

• Molecular Biology
• Biochemistry
• Gene Regulation

Background:

• Cyclin-dependent kinases (CDKs) 7, 8, and 9 are crucial for transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II.
• These kinases possess distinct roles in regulating gene expression.

Purpose of the Study:

• To investigate the hypothesis that CDK7, CDK8, and CDK9 exhibit differential substrate specificities for the CTD.
• To understand how these kinases interact with specific CTD repeats and phosphorylation states.

Main Methods:

• Utilized synthetic CTD substrates with varying heptad repeats to assess kinase preferences.
• Employed glutathione S-transferase (GST) fusion proteins to analyze CTD region phosphorylation.
• Tested the phosphorylation of a synthetic CTD peptide containing Ser-2-PO(4) by CDK complexes.

Main Results:

• CDK7, CDK8, and CDK9 demonstrated distinct preferences for phosphorylating specific heptad repeats within a synthetic CTD substrate.
• Kinases showed differential phosphorylation of CTD regions in GST fusion proteins.
• A synthetic CTD peptide with Ser-2-PO(4) was a poor substrate for CDK9 complexes.
• Binding of viral activators E1A and Tat to CDK8 and CDK9, respectively, did not alter their substrate preferences.

Conclusions:

• The phosphorylation patterns of CTD heptads by CDK7, CDK8, and CDK9 are distinct.
• The phosphorylation state and display of CTD heptads influence their recognition and phosphorylation by transcription-associated CDKs.
• These findings provide insights into the nuanced regulation of transcription by CDKs.