RADICAL MUTATIONS REVEAL TATA-BOX BINDING-PROTEIN SURFACES REQUIRED FOR ACTIVATED TRANSCRIPTION IN-VIVO

Regions on the surface of human TATA-box binding protein (TBP) require d for activated transcription in vivo were defined by construction of a library of 89 surface residue mutants with radical substitutions tha t were assayed for their ability to support activated transcription in vivo, basal transcription in vitro, and TFIIA and TFIIB binding in vi tro. four epitopes were identified in which substitutions in two to fo ur neighboring surface residues greatly inhibited activated transcript ion in vivo. One epitope in which substitutions inhibited both basal a nd activated transcription (E284, L287) is the interface between TBP a nd TFIIB. Another (A184, N189, E191, R205) is the recently determined interface between TBP and TFIIA. Mutations in residues in this TFIIA i nterface greatly inhibit activated, but not basal transcription, demon strating a requirement for the TFIIA-TBP interaction for activated tra nscription in vivo in mammalian cells. The remaining two activation ep itopes (TBP helix 2 residues R231, R235, R238, plus F250; and G175, C1 76, P247) are probably interfaces with other proteins required for act ivated transcription. The library of mutants responded virtually ident ically to two different types of activators, GL4-E1A and GAL4-VP16, in dicating that transcriptional activation by different classes of activ ators requires common interactions with TBP.