title: NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.
abstract: The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure. The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography. Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element. They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity. Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion. To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B. Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream. The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues. This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.
authors.affiliation: Groupe de RMN, DCSO, Ecole Polytechnique, F91128 Palaiseau, France.
authors.forename: A L
authors.initials: AL
authors.lastname: Vuidepot
authors.forename: F
authors.initials: F
authors.lastname: Bontems
authors.forename: M
authors.initials: M
authors.lastname: Gervais
authors.forename: B
authors.initials: B
authors.lastname: Guiard
authors.forename: E
authors.initials: E
authors.lastname: Shechter
authors.forename: J Y
authors.initials: JY
authors.lastname: Lallemand
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c; D004247:DNA; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D013941:Thymine / Q000737:chemistry*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D018911:Trinucleotide Repeats; D015032:Zinc / Q000737:chemistry*
chemical_list: C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D045304:Cytochromes c; D004247:DNA; D015032:Zinc; D013941:Thymine