title: The basis for evolution of DNA-binding specificity of the Aft1 transcription factor in yeasts.
abstract: The Saccharomyces cerevisiae Aft1 and Kluyveromyces lactis KlAft are orthologous yeast transcription activators that regulate the expression of the same group of iron-uptake genes but bind to the different DNA sites: TGCACCC for Aft1 and PuCACCC for KlAft. To establish whether the DNA-binding mechanisms of Aft1 and KlAft have diverged during the evolution of the Aft-type transcription factor, we examined the function of a nonconserved region in their DNA-binding domains. A large part of this region is composed of a sequence predicted to be disordered in structure and potentially phosphorylated. We show with deletion mutant analyses that this sequence is essential for the binding of Aft1 to its DNA site and for the iron uptake and growth of S. cerevisiae under iron-limited conditions. We constructed hybrid proteins by exchanging the nonconserved regions of Aft1 and KlAft. We show that the Aft1 region is necessary and sufficient for KlAft to bind efficiently to the Aft1 DNA site in S. cerevisiae and to complement the iron-dependent phenotype of the aft1Δaft2Δ mutant. This demonstrates that the changes in the nonconserved region of the Aft-type DNA-binding domain have led to changes in the DNA-binding specificity and have major consequences for the regulation of iron homeostasis. The combination of bioinformatic and experimental analyses indicates that the sequence TGCACCC is the most probable ancestral Aft-type element. Our findings suggest that the changes in the nonconserved region of the DNA-binding domain are responsible for the evolution of the TGCACCC sequence toward PuCACCC in the K. lactis species.
authors.affiliation: Microbiologie, Adaptation et Pathogénie, Unité Mixte de Recherche 5240, Centre National de la Recherche Scientifique, Université Lyon 1, Bayer CropScience, F-69622 Villeurbanne, France.
authors.forename: Isabelle R
authors.initials: IR
authors.lastname: Gonçalves
authors.forename: Natalia
authors.initials: N
authors.lastname: Conde e Silva
authors.forename: Cesar La Torre
authors.initials: CL
authors.lastname: Garay
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Lesuisse
authors.forename: Jean Michel
authors.initials: JM
authors.lastname: Camadro
authors.forename: Pierre Louis
authors.initials: PL
authors.lastname: Blaiseau
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D007501:Iron / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: C090297:AFT1 protein, S cerevisiae; C436328:Aft2 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D007501:Iron
keywords: Aft-type regulator; Kluyveromyces lactis; Saccharomyces cerevisiae; electrophoretic mobility shift assays; iron homeostasis