Determination of the Neurospora crassa CYS 3 sulfur regulatory protein consensus DNA-binding site: amino-acid substitutions in the CYS3 bZIP domain that alter DNA-binding specificity.
CYS3 is the positive-acting global regulatory protein involved in the sulfur control circuit in Neurospora crassa and belongs to the family of bZIP DNA-binding proteins. Here we report a characterization of native DNA-binding sites recognized by CYS3. DNA footprinting experiments and systematic mutational analysis were used to define the consensus CYS3-binding sequence, 5'-ATGPuPyPuPyCAT, a 10-bp palindrome. The sequence 5'-ATGACGTCAT acts as a strong binding site, and all single nucleotide changes within this sequence resulted in a reduction, or even complete loss, of CYS3 DNA-binding. Site-directed mutagenesis was employed to study two uncharged residues, serine 113 and phenylalanine 116, in the basic region of the CYS3 protein bZip DNA-binding domain. Ser113 appears to be directly involved in a specific interaction with nucleotide 2 of the binding site, possibly by making a direct contact with this base, and Phe116 contributes significantly to DNA-binding affinity.
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Department of Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA.
Marzluf
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D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003542:Cystathionine gamma-Lyase; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009492:Neurospora crassa / Q000737:chemistry*; D010649:Phenylalanine / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase