THE DNA-BINDING DOMAIN OF THE YEAST SACCHAROMYCES-CEREVISIAE CYP1(HAP1) TRANSCRIPTION FACTOR POSSESSES 2 ZINC IONS WHICH ARE COMPLEXED IN AZINC CLUSTER

Various fragments of the N-terminal, DNA-binding domain of the yeast S accharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypepti des have been analysed biochemically and we have undertaken a more ext ensive physical study of a fragment consisting of amino acids 49-126 [ CYP1(49-126)]. We show that this CYP1(49-126) peptide requires zinc or cadmium in the growth medium in order to maintain a stable structure. A method to purify CYP1(49-126) is presented. We demonstrate that the purified CYP1(49-126) fragment contains two zinc ions/fragment or two cadmium ions/fragment, which are necessary for DNA binding. Cd-113 on e-dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coord ination, and that it forms a zinc-cluster complex Like GALA.