GAL4 INTERACTS WITH TATA-BINDING PROTEIN AND COACTIVATORS

A major goal in understanding eukaryotic gene regulation is to identif y the target(s) of transcriptional activators. Efforts to date have po inted to various candidates. Here we show that a 34-amino-acid peptide from the carboxy terminus of GAL4 is a strong activation domain (AD) and retains at least four proteins from a crude extract: the negative regulator GAL80, the TATA-binding protein (TBP), and the putative coac tivators SUG1 and ADA2. TFIIB was not retained. Concentrating on TBP, we demonstrate in in vitro binding assays that its interaction with th e AD is specific, direct, and salt stable up to at least 1.6 M NaCl. T he effects of mutations in the GALA AD on transcriptional activation i n vivo correlate with their affinities to TBP. A point mutation (L114K ) in yeast TBP, which has been shown to compromise the mutant protein in both binding to the VP16 AD domain and activated transcription in v itro, reduces the affinity to the GAL4 AD to the same degree as to the VP16 AD. This suggests that these two prototypic activators make simi lar contacts with TBP.