title: Mutations in the basic loop of the Zn binuclear cluster of the UaY transcriptional activator suppress mutations in the dimerisation domain.
abstract: UaY is the specific ZnII(2)Cys(6) transcriptional activator of the purine utilisation pathway in Aspergillus nidulans. Previous work has determined the consensus binding sequence by EMSA and foot-printing. We determine here that it binds as a dimer to its cognate CGG-N(6)-CCG sites. We identify the uaY109 mutation, which has been shown to affect differentially the expression of a number of UaY-regulated genes, as a F112I substitution in the DNA-binding motif dimerisation domain. We isolated back mutants, revertants carrying different residues at the same position (I112N and I112M) and intragenic suppressors mapping in the first loop of the Zn cluster (N75T and N75K). We have analysed the original mutant and its revertants by growth tests and by their effects on the mRNA steady states of five UaY-regulated genes. We have determined the effect of the different mutations on UaY dimerisation, on the apparent Kdiss of the UaY DNA-binding domain to appropriate DNA sequences and on the methylation interference pattern. We have attempted to rationalise these phenotypes by modelling the UaY DNA binding domain on the structure of the highly similar Ppr1p. However, modelling of the wild-type and mutant proteins provides only a partial explanation for the observed phenotypes. This suggests that the mutated residues may have other roles besides the obvious ones inferred from their position in the sequence and by the similarity of UaY and Ppr1p.
authors: Microbiología, Instituto de Química Biológica, Universidad de la República, Casilla de Correo 1157, Montevideo, Uruguay. gianna.cecchetto@gmail.com
authors: Gianna
authors: G
authors: Cecchetto
authors: Mariana
authors: M
authors: Richero
authors: Nathalie
authors: N
authors: Oestreicher
authors: María Isabel
authors: MI
authors: Muro-Pastor
authors: Sergio
authors: S
authors: Pantano
authors: Claudio
authors: C
authors: Scazzocchio
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D015536:Down-Regulation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D015534:Trans-Activators