The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding.
The E2 transactivator of bovine papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID.
Unité des Virus Oncogénes, URA 1644 du CNRS, Institut Pasteur, Paris, France.
G
G
Steger
J
J
Ham
O
O
Lefebvre
M
M
Yaniv
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D009838:Oligodeoxyribonucleotides; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D014764:Viral Proteins