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

The DNA binding domain of the yeast transcriptional activator CYP1(HAP 1) contains a zinc-cluster structure, The structures of the DNA bindin g domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PP R1) have been studied by X-ray crystallography, Their binding domains present, besides the zinc: cluster, a short linker peptide and a dimer ization element, They recognize, as homodimers, two rotationally symme tric CGG trinucleotides, the linker peptide and the dimerization eleme nt playing a crucial role in binding specificity, Surprisingly, CYP1 r ecognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion, To better understand the b inding specificity of CYP1, we have studied, by NMR, the interaction b etween 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, a s for GAL4 and PPR1, and the thymine is bound in the minor groove by t he N-terminal region, which possesses a basic stretch of arginyl and l ysyl residues. This suggests that the CYP1(55-126) N-terminal region c ould play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1.