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J. Biol. Chem., Vol. 276, Issue 19, 15886-15892, May 11, 2001

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TAF_II 250 Phosphorylates Human Transcription Factor IIA on Serine Residues Important for TBP Binding and Transcription Activity^*

Steven Solow, Moreh Salunek, Robert Ryan, and Paul M. Lieberman^§

From the Wistar Institute, Philadelphia, Pennsylvania 19104

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 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_II250) phosphorylated TFIIA on the  subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF_II250 phosphorylation in vitro. The NH₂-terminal kinase domain of TAF_II250 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_II250 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  subunit Y65A mutation strongly stimulated TFIIA·TBP·TATA complex formation. TFIIA-Y65A is defective for binding to the -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.

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