| The positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is composed of the cyclin dependent kinase, Cdk9, and a cyclin partner, T1, T2a, T2b or K. In mammalian cells there are two forms of P-TEFb, a small kinase-active form and an inactive P-TEFb complex containing the 7SK snRNA and the HEXIM1 protein. Using a defined system, it was found that HEXIM1 and 7SK are both necessary and sufficient for inhibition of P-TEFb. Furthermore, homologs of HEXIM1 and 7SK were identified in Drosophila melanogaster and subsequently demonstrated to be components of a larger molecular weight P-TEFb complex that releases P-TEFb when higher activity levels are required.;Using HIV as a model system because it requires Tat to recruit P-TEFb, it was found that transfection of Tat caused a dramatic shift of P-TEFb out of the snRNP to a smaller form containing Tat. In vitro, Tat blocked the formation of the P-TEFb·HEXIM1·7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb·HEXIM1·7SK complex, and, in vivo, a P-TEFb-binding Tat mutant was unable to cause this release. These results demonstrate that a transcriptional activator can acquire P-TEFb from the 7SK snRNP by binding to P-TEFb. It was also found that HEXIM1 binds to the HIV 5' UTR (TAR) and recruits and inhibits P-TEFb activity. Importantly, HEXIM1 was found by ChIP to be present at the HIV LTR promoter. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.;To better understand the biochemical mechanisms involved in the inhibition of P-TEFb by HEXIM and RNA, and the required activation through release, studies have been initiated to determine the x-ray crystal structures. Several Cyclin T1, Cdk9, and HIV Tat constructs have been designed, cloned, the complexes co-expressed and purified. Following biochemical characterization of these protein, crystallization trials have been successful in forming high quality P-TEFb·Tat crystals and low quality P-TEFb·Tat·TAR crystals. Additionally, several HEXIM1 constructs were cloned, expressed, and purified, and are currently in crystal trials with TAR. |
