The DNA-binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster.
Various fragments of the N-terminal, DNA-binding domain of the yeast Saccharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypeptides have been analysed biochemically and we have undertaken a more extensive 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. 113Cd one-dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coordination, and that it forms a zinc-cluster complex like GAL4.
Département de Chimie de Synthèse Organique, Ecole Polytechnique, Palaiseau, France.
J E
JE
Timmerman
B
B
Guiard
E
E
Shechter
M A
MA
Delsuc
J Y
JY
Lallemand
M
M
Gervais
D000595:Amino Acid Sequence; D001483:Base Sequence; D002104:Cadmium / Q000737:chemistry; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013054:Spectrophotometry, Atomic; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors; D015032:Zinc / Q000737:chemistry*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D002104:Cadmium; D015032:Zinc