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) required for activated transcription in vivo were defined by construction of a library of 89 surface residue mutants with radical substitutions that were assayed for their ability to support activated transcription in vivo, basal transcription in vitro, and TFIIA and TFIIB binding in vitro. four epitopes were identified in which substitutions in two to four neighboring surface residues greatly inhibited activated transcription in vivo. One epitope in which substitutions inhibited both basal and activated transcription (E284, L287) is the interface between TBP and TFIIB. Another (A184, N189, E191, R205) is the recently determined interface between TBP and TFIIA. Mutations in residues in this TFIIA interface greatly inhibit activated, but not basal transcription, demonstrating a requirement for the TFIIA-TBP interaction for activated transcription in vivo in mammalian cells. The remaining two activation epitopes (TBP helix 2 residues R231, R235, R238, plus F250; and G175, C176, P247) are probably interfaces with other proteins required for activated transcription. The library of mutants responded virtually identically to two different types of activators, GL4-E1A and GAL4-VP16, indicating that transcriptional activation by different classes of activators requires common interactions with TBP.