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 express ion by increasing both the rate of transcription initiation and the ef ficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that com ponents of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with t he human TATA-binding protein (TBP) and specific TBP-associated factor s (TAFs) that comprise the TFIID complex. Here, in vitro glutathione S -transferase protein binding assays containing fully functional or tra nsactivation-defective mutant Tat proteins have been used to investiga te the functional significance of the direct interaction between Tat a nd 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 fai l to inhibit Tat-TBP binding. Full-length Tat, the activation domain o f Tat alone, and a transactivation-defective mutant of Tac that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TB P provided that the H1 alpha helical domain that maps to amino acids 1 67-220 within the highly conserved carboxyl terminus of TBP is maintai ned. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding, is required fo r transactivation by Tat.