ASSOCIATION OF TAT WITH PURIFIED HIV-1 AND HIV-2 TRANSCRIPTION PREINITIATION COMPLEXES

The HIV-1 (human immunodeficiency virus type 1) and HIV-2 Tat proteins increase the level of transcription from their corresponding long ter minal repeats. Tat activates transcription likely by interaction with components of the transcriptional initiation and elongation complexes during different stages of the transcription reaction. In the current study, two approaches were used to address the sites at which Tat beco mes stably associated with the HIV transcription complex. First, we is olated column purified HIV-1 and HIV-2 transcription complexes that we re competent for in vitro transcription and found that wild-type but n ot mutant Tat protein was specifically associated with this complex. A n intact HIV TATA element and the presence of functional TATA-binding protein were necessary for Tat association. In contrast, the HIV-1 and HIV-2 TAR bulge sequences which serve as binding sites for Tat were n ot required for its association with the HIV preinitiation complex. A second complementary approach using immobilized HIV-1 and HIV-2 templa tes also demonstrated a functional association of Tat with HIV-1 and H IV-2 preinitiation complexes. Wild-type but not mutant Tat proteins as sociated with transcription complexes assembled on immobilized HIV-1 a nd HIV-2 templates and the association of Tat correlated with increase s in the level of in vitro transcription. These results indicate that Tat can associate with HIV-1 and HIV-2 transcription complexes prior t o the initiation of transcription by RNA polymerase II.