title: The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite.
abstract: Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.
authors.affiliation: Department of Molecular Genetics, Ohio State University, Columbus 43210.
authors.forename: M N
authors.initials: MN
authors.lastname: Kanaan
authors.forename: Y H
authors.initials: YH
authors.lastname: Fu
authors.forename: G A
authors.initials: GA
authors.lastname: Marzluf
mesh_terms: D000595:Amino Acid Sequence; D003542:Cystathionine gamma-Lyase; D004271:DNA, Fungal / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase