Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation.
OBJECTIVE
To examine the functional interaction between HIV-1 Tat protein and the TATA-binding protein (TBP).


METHODS
HIV long terminal repeat reporter plasmids were cotransfected into mammalian and Drosophila insect cells with expression vectors encoding Tat and vectors encoding TBP either alone or linked to an heterologous DNA-binding domain.


METHODS
The activity of the different reporters was compared in the presence or absence of Tat or TBP, or both, upon cotransfections into human and Drosophila insect cell lines.


RESULTS
Tat protein is unable to transactivate enhancerless HIV-1 minimal promoter bearing only the TATA box and TAR. Artificial recruitment of human TBP (hTBP) to the enhancerless HIV minimal promoter was found to trigger gene expression and coexpression of Tat resulted in a marked synergy. Tat protein cooperated with DNA-bound hTBP by inducing high levels of processive viral transcripts. Synergy between Tat and hTBP was also observed when both factors were targeted to a promoter DNA template. The functional cooperation between TBP and Tat was further demonstrated using the Drosophila Schneider SL2 cells. In these cells Tat protein alone was ineffective; however, coexpression of Drosophila TBP and Tat resulted in a trans-activating response region-dependent synergistic transactivation of basal transcription.


CONCLUSIONS
The strong synergy between TBP and Tat in the absence of any DNA-bound activator suggests that Tat stimulates transcription in an activator-independent manner most likely by a functional interaction with general transcription factors that occurs after TBP recruitment. Thus, efficient recruitment of TBP represents a limiting step for Tat transactivation.
Majello
B
B
Department of Genetics, Molecular and General Biology, University of Naples Federico II, Italy.
Napolitano
G
G
Lania
L
L
D000818:Animals; D002460:Cell Line; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology; D004625:Embryo, Nonmammalian / Q000166:cytology; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus