Quantitation of putative activator-target affinities predicts transcriptional activating potentials.
We quantitate the 'activating potentials' of deletion and point mutation variants of a 42 amino acid yeast transcriptional activating region excised from the yeast activator GAL4 and, using surface plasmon resonance, we measure the relative affinities of these molecules for a variety of proteins, including plausible target proteins as well as GAL80, a specific inhibitor of GAL4. We find a remarkable correlation between the relative activating potentials of the derivatives and their relative affinities for yeast TBP and for yeast TFIIB; other tested proteins interacted significantly more weakly, if at all. These results provide an especially strong argument that TBP and TFIIB are activating region targets. We also show, using one set of yeast activating region mutants, that activator-target interactions are strongly correlated with the length of the activating region, that the effect of point mutants is highly dependent on the length of the activating region mutated and that, unlike interactions with TBP and TFIIB, interaction with the specific inhibitor GAL80 is destroyed by deletion of certain critical residues in the C-terminal half of the 42 amino acid activating region.
Wu
Y
Y
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Reece
R J
RJ
Ptashne
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors