Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment.
Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain 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 functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.
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Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA. hxiao@atlas.niaid.nih.gov
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D000256:Adenoviridae / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C070633:Gal-VP16; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors