A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites.
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.
Molle
Dorothée
D
IGMM-CNRS UMR 5535, Montpellier, France. dorothee.molle@igmm.cnrs.fr
Maiuri
Paolo
P
Boireau
Stéphanie
S
Bertrand
Edouard
E
Knezevich
Anna
A
Marcello
Alessandro
A
Basyuk
Eugenia
E
D045744:Cell Line, Tumor; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010445:Peptide Elongation Factors; D012367:RNA, Viral; C020746:factor EF-P; D054322:tat Gene Products, Human Immunodeficiency Virus; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9