PROTEASE FOOTPRINTING ANALYSIS OF TERNARY COMPLEX-FORMATION BY HUMAN TFIIA

Authors
Citation
R. Hori et M. Carey, PROTEASE FOOTPRINTING ANALYSIS OF TERNARY COMPLEX-FORMATION BY HUMAN TFIIA, The Journal of biological chemistry, 272(2), 1997, pp. 1180-1187
Citations number
49
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
272
Issue
2
Year of publication
1997
Pages
1180 - 1187
Database
ISI
SICI code
0021-9258(1997)272:2<1180:PFAOTC>2.0.ZU;2-9
Abstract
Transcription factor (TF) IIA performs two important regulatory functi ons during RNA polymerase II transcription: it is required for efficie nt binding of TFIID to a core promoter and it mediates the effects of upstream activators, both through direct interaction with the TATA box binding protein (TBP). To begin studying how TFIIA mediates these eff ects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA . TBP . TATA t ernary complex formation. Chymotrypsin and proteinase K cleavage patte rns of TFIIA bearing a P-32-end-labeled gamma subunit revealed that am ino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone. I n contrast, amino acids 341-367 in the beta portion of a P-32-labeled alpha-beta subunit were protected in the ternary but not in the binary complex, implying that those residues interact with promoter DNA. The regions of human TFIIA identified by protease footprinting are homolo gous to and encompass the yeast TFIIA residues that contact TBP and DN A in the recently solved crystal structure of the yeast ternary comple x. The conservation of the regions and residues mediating complex form ation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter.