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
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.