Identification of highly conserved amino-terminal segments of dTAF(II)230 and yTAF(II)145 that are functionally interchangeable for inhibiting TBP-DNA interactions in vitro and in promoting yeast cell growth in vivo

Abstract

TFIID is a multiprotein complex composed of TBP and several TAF(II)s. Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAF(II)230 (dTAF(II)230) and yeast TAF(II)145 (yTAF(II)145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAF(II)145 TAND (yTAND) was divided into two subdomains, yTANDI10-37 and yTANDII46-71, that function cooperatively. Here, we identify dTANDII within the amino terminus of dTAF(II)230 at 118-143 amino acids in addition to dTANDI18-77, reported previously, dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast strains containing mutant yTAF(II)145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 °C. In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAF(II)145- TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.