Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo
Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo
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DOI：
10.1101/gad.10.19.2491
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发表时间：
1996-10-01
影响因子：
10.5
通讯作者：
Berk, AJ
中科院分区：
文献类型：
--
作者：
Bryant, GO;Martel, LS;Berk, AJ
通过构建含有89个表面残基突变体的文库来确定人TATA盒结合蛋白（TBP）表面上体内激活转录所需的区域，所述表面残基突变体具有自由基取代，测定其支持体内激活转录、体外基础转录以及体外TFIIA和TFIIB结合的能力。鉴定了四个表位，其中两个至四个相邻表面残基的取代极大地抑制了体内激活的转录。其中取代抑制基础和活化转录的一个表位（E284，L287）是TBP和TFIIB之间的界面。另一个（A184、N189、E191、R205）是最近确定的TBP与TFIIA之间的界面。该TFIIA界面中残基的突变极大地抑制了激活转录，但不抑制基础转录，这表明哺乳动物细胞体内激活转录需要TFIIA-TBP相互作用。剩余的两个活化表位（TBP螺旋2残基R231、R235、R238加上F250;和G175、C176、P247）可能与活化转录所需的其他蛋白质相互作用。突变体库对两种不同类型的激活剂GL 4-E1 A和GAL 4-VP 16的反应几乎相同，表明不同类型的激活剂的转录激活需要与TBP的共同相互作用。
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.