PROMOTER ACTIVITY OF TAT AT STEPS SUBSEQUENT TO TATA-BINDING PROTEIN RECRUITMENT

Artificial recruitment of TATA-binding protein (TBP) to many eukaryoti c promoters bypasses DNA-bound activator function. The human immunodef iciency virus type 1 (HIV-1) Tat is an unconventional activator that u p-regulates transcription from the HIV-1 long terminal repeat (LTR) th rough binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA p olymerase II (RNAP II) transcriptional unit, the precise limiting step s for HIV-1 transcription and how Tat resolves these limitations remai n incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting-step in HIV-1 LTR transcription and whether Tat func tions to recruit TBP. As a control, we compared the activity of the ad enovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievab le with the potent GAL4-VP16 activator. By contrast, TBP recruitment t o the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcr iption. These results document that the HIV-1 and the E1b promoters ar e transcriptionally limited at different steps; the major rate-limitin g step for E1b is recruitment of TBP, while activation of the HIV-1 LT R requires steps in addition to TBP recruitment. We suggest that Tat a cts to accelerate rate-limiting steps after TBP recruitment.