Transcription factor IIA (
TFIIA) is a positive acting general factor that contacts the
TATA-binding protein (
TBP) and mediates an activator-induced conformational change in the
transcription factor IID (
TFIID) complex. Previously, we have found that phosphorylation of yeast
TFIIA stimulates
TFIIA.
TBP.TATA complex formation and transcription activation in vivo. We now show that human
TFIIA is phosphorylated in vivo on
serine residues that are partially conserved between yeast and human
TFIIA large subunits.
Alanine substitution mutation of
serine residues 316 and 321 in
TFIIA alphabeta reduced
TFIIA phosphorylation significantly in vivo. Additional
alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four
serine residues reduced the ability of
TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that
TFIIA phosphorylation is required globally for optimal function. In vitro, holo-
TFIID and
TBP-associated factor 250 (TAF(II)250) phosphorylated
TFIIA on the beta subunit. Mutation of the four serines required for in vivo phosphorylation eliminated
TFIID and TAF(II)250 phosphorylation in vitro. The NH(2)-terminal
kinase domain of TAF(II)250 was sufficient for
TFIIA phosphorylation, and this activity was inhibited by full-length
retinoblastoma protein but not by a
retinoblastoma protein mutant defective for TAF(II)250 interaction or
tumor suppressor activity.
TFIIA phosphorylation had little effect on the
TFIIA.
TBP.TATA complex in electrophoretic mobility shift assay. However, phosphorylation of
TFIIA containing a gamma subunit Y65A mutation strongly stimulated
TFIIA.
TBP.TATA complex formation. TFIIA-gammaY65A is defective for binding to the beta-sheet domain of
TBP identified in the crystal structure. These results suggest that
TFIIA phosphorylation is important for strengthening the
TFIIA.
TBP contact or creating a second contact between
TFIIA and
TBP that was not visible in the crystal structure.