In vitro interaction of the human immunodeficiency virus type 1 Tat transactivator and the general transcription factor TFIIB with the cellular protein TAP.
We have reported the molecular cloning, expression, and characterization of a human cellular protein, TAP, which possesses a strong transcriptional activation domain and binds the human immunodeficiency virus type 1 Tat transactivator in vitro and in vivo (L. Yu, Z. Zhang, P.M. Loewenstein, K. Desai, Q. Tang, D. Mao, J.S. Symington, and M. Green, J. Virol. 69:3007-3016, 1995). Here we show that TAP binds the general transcription factor TFIIB. Furthermore, we delineate the binding domains of TAP, Tat, and TFIIB, as well as measure the strengths and specificity of these protein-protein interactions. TAP binds strongly to Tat, with a Kd of (approximately 2 to 5) x 10(-7) M. The Tat activation region contains a 17-amino-acid conserved core domain which is the single contact site for TAP. Single-amino-acid substitutions within the Tat core domain inactivate transactivation in vivo and in vitro and greatly reduce binding of Tat to TAP in vitro. TAP binds strongly to TFIIB, with about the same Kd as for Tat. The interaction between TAP and TFIIB requires a sequence near the carboxy terminus of TFIIB which is also required for binding the strong acidic activator VP16. The contact sites for Tat and TFIIB map within the TAP C-terminal region, which contains the TAP activation domain. These combined results are consistent with the hypothesis that TAP is a cellular coactivator that bridges the Tat transactivator to the general transcription machinery via TFIIB.
Yu
L
L
Institute for Molecular Virology, Saint Louis University School of Medicine, Missouri 63110, USA.
Loewenstein
P M
PM
Zhang
Z
Z
Green
M
M
D000074183:ATPases Associated with Diverse Cellular Activities; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D011994:Recombinant Proteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D046988:Proteasome Endopeptidase Complex; D000074183:ATPases Associated with Diverse Cellular Activities; C064757:PSMC3 protein, human