Protease footprinting analysis of ternary complex formation by human TFIIA.
Transcription factor (TF) IIA performs two important regulatory functions during RNA polymerase II transcription: it is required for efficient 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 effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA x TBP x TATA ternary complex formation. Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a 32P-end-labeled gamma subunit revealed that amino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone. In contrast, amino acids 341-367 in the beta portion of a 32P-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 homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of the yeast ternary complex. The conservation of the regions and residues mediating complex formation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter.
Hori
R
R
Department of Biological Chemistry, University of California, Los Angeles 90095-1737, USA.
Carey
M
M
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010449:Peptide Mapping / Q000379:methods*; D011487:Protein Conformation; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D004247:DNA; D010450:Endopeptidases