Structural studies on P-TEFb and its role in regulation of transcription elongation

Transcription is the process during which genes are transcribed into messenger RNA that in turn serves as the template for protein synthesis. Regulation of transcription ensures the timely expression of proteins required for cell growth and differentiation. Errors in transcription regulation can lead to uncontrolled cell growth and proliferation. Positive transcription elongation factor b (P-TEFb) controls the elongation phase of transcription that is carried out by RNA polymerase II. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, it is also a critical host cellular cofactor for the human immunodeficiency virus HIV. HIV uses P-TEFb to enable the transcription of its own genome by the cellular transcription apparatus. Increases in P-TEFb activity are also associated with other diseases, for example cardiac hypertrophy and breast cancer. Our study aims not only to contribute to a better understanding of how P-TEFb recognizes its substrates and how its activity is regulated within the cell, but also to identify the mode of action of specific inhibitors of P-TEFb. One such inhibitor is Flavopiridol, a potential drug against cancer that is already in clinical trials. These studies will form the basis for future structure-aided drug discovery initiatives to develop therapeutics targeting P-TEFb. Within the cell P-TEFb activity is controlled by its association with positive and negative regulatory factors, called BRD4 and HEXIM1/7SK RNA, respectively. Our knowledge of the mechanisms by which these regulatory factors control P-TEFb activity would be greatly assisted by knowing their structures in molecular detail. Protein structures can be determined using the techniques of X-ray crystallography and electron microscopy. The aim of the proposed research is to use both these techniques to characterise the molecular details of the active site as well as the organization of the large inhibitory complex. This information will be useful in furthering our understanding of how transcriptional activity is controlled at the stage of transcription elongation and will also facilitate our understanding of how HIV uses the P-TEFb to promote transcription of its own genes. Ultimately we may be able to exploit this knowledge for the treatment of disease.

In eukaryotes control of transcription is critical for the timely expression of genes during cell growth and differentiation. The different stages of transcription initiation, elongation, 3?end formation and termination, are tightly regulated by different factors which assemble on the 52-repeat C-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II). Patterns of CTD phosphorylation and dephosphorylation change during the transcription cycle, providing a dynamic assembly platform for the appropriate proteins. Positive transcription elongation factor b (P-TEFb) is a cyclin dependent kinase, CDK9, which phosphorylates the CTD as well as negative elongation factors and thereby promotes processive transcription elongation. Brd4 and HEXIM1/7SK RNA, are respectively positive and negative regulators that dynamically control P-TEFb activity. While Brd4 recruits P-TEFb to sites of active transcription via its bromo domains, association with HEXIM1/7SK RNA leads to the formation of an inactive RNA/protein complex. Association with Brd4 or HEXIM1/7SK RNA is mutually exclusive and stress treatment causes a conversion of the inactive HEXIM1/7SK RNA to the active Brd4-bound form of P-TEFb. The functional equilibrium between the active and inactive forms is disturbed in several major human diseases, such as cardiac hypertrophy, breast cancer and Human immunodeficiency virus (HIV) infection. HIV type 1 uses P-TEFb as a specific cofactor for efficient transcriptional elongation of its genome. To provide a greater understanding of the molecular interactions that mediate P-TEFb substrate selectivity we have recently solved the structure of the CDK9/cyclinT sub-complex. We plan to use SPR and ITC to analyse the interaction of P-TEFb with its substrates and use the insights from these studies to identify CDK9/cyclinT/substrate complexes for crystallisation and structural analysis. The involvement of P-TEFb in cardiac hypertrophy, breast cancer and HIV make it a possible drug target for the treatment of these diseases. Determination of structures for P-TEFb in complex with the clinically relevant CDK9 inhibitor Flavopiridol, and DRB (5,6-dichloro-1-b-D-ribufuranosylbenzimidazole) will allow us to elaborate the binding modes of potent CDK9 inhibitors within the ATP binding site. The second part of the project will address the mechanism by which the natural CDK9 inhibitor HEXIM1/7SK RNA inhibits P-TEFb. A combination of electron microscopy and X-ray crystallography will be used to tackle this complex structural challenge and we will elucidate the relationships between structure and function within the P-TEFb/HEXIM1/7SK RNA complex by in vitro transcription assays.