Phosphorylation of TFIIA stimulates TATA binding protein-TATA interaction and contributes to maximal transcription and viability in yeast

Posttranslational modification of general transcription factors may be an i mportant mechanism for global gene regulation, The general transcription fa ctor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential f or high-level transcription mediated by various activators. Modulation of t he TFIIA-TBP interaction is a likely target of transcriptional regulation, We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylat ed in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA compl ex and is required for high-level transcription. Alanine substitution of se rine residues 220, 225, and 232 completely eliminated in vivo phosphorylati on of Toa1, although no single amino acid substitution of these serine resi dues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold m ore efficient in forming a stable complex with TBP and TATA DNA. Dephosphor ylation of yeast-derived TFIIA reduced DNA binding activity, and recombinan t TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing the teal S220/225/232A showed reduced high-lev el transcriptional activity at the URA1, URA3, and HIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined,vith an alanine substitution mutation at W285, which disrupts the TFIIA-TBP inte rface. Phosphorylation of TFIIA could therefore be an important mechanism o f transcription modulation, since it stimulates TFIIA-TBP association, enha nces high-level transcription, and contributes to yeast viability.