The ETS family member ERM contains an alpha-helical acidic activation domain that contacts TAFII60.
Transcription factors are modular entities built up of discrete domains, some devoted to DNA binding and others permitting transcriptional modulation. The structure of DNA binding domains has been thoroughly investigated and structural classes clearly defined. In sharp contrast, the structural constraints put on transactivating regions, if any, are mostly unknown. Our investigations focus on ERM, a eukaryotic transcription factor of the ETS family. We have previously shown that ERM harbours two transactivating domains (TADs) with distinct functional features: AD1 lies in the first 72 amino acids of ERM, while AD2 sits in the last 62. Here we show that AD1 is a bona fide acidic TAD, for it activated transcription in yeast cells, while AD2 did not. AD1 contains a 20 amino acid stretch predicted to form an alpha-helix that is found unchanged in the related PEA3 and ER81 transcription factors. Circular dichroism analysis revealed that a 32 amino acid peptide encompassing this region is unstructured in water but folds into a helix when the hydrophobic solvent trifluoroethanol is added. The isolated helix was sufficient to activate transcription and mutations predicted to disrupt it dramatically affected AD1-driven transactivation, whereas mutations decreasing its acidity had more gentle effects. A phenylalanine residue within the helix was particularly sensitive to mutations. Finally, we observed that ERM bound TAFII60 via AD1 and bound TBP and TAFII40, presumably via other activation domains.
Defossez
P A
PA
Mécanismes du Développement et de la Cancérisation, UMR 319 CNRS/Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue Calmette, BP 447, 59021 Lille Cedex, France.
Baert
J L
JL
Monnot
M
M
de Launoit
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators / Q000378:metabolism; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C086680:ETV5 protein, human; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C084120:e(y)1 protein, Drosophila