The regulation and inhibition of P-TEFb

Thesis

Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy.

In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors.

In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.

Authors: Hole, A

• Publication date: 2011
• DOI: 10.5287/ora-44nee9704
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: phosphorylation; transcription; cyclin dependent kinase
• Subjects: Molecular biophysics (biochemistry); Biochemistry