Wild-type and transactivation-defective mutants of human immunodeficiency virus type 1 Tat protein bind human TATA-binding protein in vitro.
Tat regulates human immunodeficiency virus type 1 (HIV-1) gene expression by increasing both the rate of transcription initiation and the efficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that components of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with the human TATA-binding protein (TBP) and specific TBP-associated factors (TAFS) that comprise the TFIID complex. Here, in vitro glutathione S-transferase protein binding assays containing fully functional or transactivation-defective mutant Tat proteins have been used to investigate the functional significance of the direct interaction between Tat and TBP relative to Tat transactivation. Results demonstrate that full-length Tat, as well as the activation domain of Tat alone, binds human TBP in vitro. Site-directed mutations within the activation domain of Tat (C22G and P18IS) that abrogate transactivation by Tat in vivo fail to inhibit Tat-TBP binding. Full-length Tat, the activation domain of Tat alone, and a transactivation-defective mutant of Tat that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TBP provided that the H1 alpha helical domain that maps to amino acids 167-220 within the highly conserved carboxyl terminus of TBP is maintained. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding. is required for transactivation by Tat.
Wang
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Z
Department of Pathology and Laboratory Medicine, Tulane Cancer Center, Tulane University Medical Center, New Orleans, USA.
Morris
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GF
Rice
A P
AP
Xiong
W
W
Morris
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CB
D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D005982:Glutathione Transferase