HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation.
A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites
Molle, D.; Maiuri, P.; Boireau, S.; Bertrand, E.; Knezevich, A.; Marcello, A.; Basyuk, E.
2007 / vol 4 / pages 36
IGMM team(s) involved in this publication
Biogenèse des ARNs
Cell Line, Tumor; Humans; *Transcription, Genetic; Protein Binding; Kinetics; Cyclin-Dependent Kinase 9/metabolism; tat Gene Products, Human Immunodeficiency Virus; *HIV Long Terminal Repeat; Trans-Activation (Genetics); Fluorescence Recovery After Photobleaching; Gene Products, tat/*metabolism; HIV-1/*genetics/physiology; Peptide Elongation Factors/*metabolism; RNA, Viral/*metabolism