The regulation and inhibition of P-TEFb
<p>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. Shor...
| Glavni autor: | |
|---|---|
| Daljnji autori: | |
| Format: | Disertacija |
| Jezik: | English |
| Izdano: |
2011
|
| Teme: |
| _version_ | 1826315773041180672 |
|---|---|
| author | Hole, A |
| author2 | Noble, M |
| author_facet | Noble, M Hole, A |
| author_sort | Hole, A |
| collection | OXFORD |
| description | <p>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.</p><p>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.</p><p>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.</p> |
| first_indexed | 2024-03-06T19:16:56Z |
| format | Thesis |
| id | oxford-uuid:18ba1399-e3db-4ed6-bd9b-019bbd3c0b65 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:32:11Z |
| publishDate | 2011 |
| record_format | dspace |
| spelling | oxford-uuid:18ba1399-e3db-4ed6-bd9b-019bbd3c0b652024-12-01T15:28:46ZThe regulation and inhibition of P-TEFbThesishttp://purl.org/coar/resource_type/c_db06uuid:18ba1399-e3db-4ed6-bd9b-019bbd3c0b65Molecular biophysics (biochemistry)BiochemistryEnglishOxford University Research Archive - Valet2011Hole, ANoble, MEndicott, J<p>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.</p><p>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.</p><p>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.</p> |
| spellingShingle | Molecular biophysics (biochemistry) Biochemistry Hole, A The regulation and inhibition of P-TEFb |
| title | The regulation and inhibition of P-TEFb |
| title_full | The regulation and inhibition of P-TEFb |
| title_fullStr | The regulation and inhibition of P-TEFb |
| title_full_unstemmed | The regulation and inhibition of P-TEFb |
| title_short | The regulation and inhibition of P-TEFb |
| title_sort | regulation and inhibition of p tefb |
| topic | Molecular biophysics (biochemistry) Biochemistry |
| work_keys_str_mv | AT holea theregulationandinhibitionofptefb AT holea regulationandinhibitionofptefb |