THE BOVINE PAPILLOMAVIRUS-1 E2-PROTEIN CONTAINS 2 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 act ivate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TA TA binding protein (TBP) or by the addition of either SP1 binding site s or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. S urprisingly, purified E2 and TBP bind co-operatively to DNA in vitro w hen the sites are closely spaced. E2 does not affect the on rate of as sociation but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transact ivation and DNA binding domains. We demonstrate that the TBP stabilizi ng 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 eviden t in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcri ption of the MLP in vitro, but does not activate promoters with SP1 si tes. In conclusion, our results show that the E2 transactivation domai n has a modular structure. We have identified one domain which probabl y 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 i n vitro. The classic N-terminal activation domain of E2 might affect o ne or several step(s) in the assembly of the preinitiation complex occ urring after the binding of TFIID.