Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors.
Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.
Pinson
B
B
Institut de Biochimie et Génétique Cellulaires, CNRS UMR5095, 1 Rue Camille Saint-Saëns, F-33077 Bordeaux Cedex, France. benoit.pinson@ibgc.u-bordeaux2.fr
Brendeford
E M
EM
Gabrielsen
O S
OS
Daignan-Fornier
B
B
D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020570:Genes, myb; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D020626:Oncogene Proteins v-myb / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
C061767:BAS1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020626:Oncogene Proteins v-myb; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; D003545:Cysteine