title: Analysis of the sulfur-regulated control of the cystathionine γ-lyase gene of Neurospora crassa.
abstract: BACKGROUND
  Cystathionine γ-lyase plays a key role in the transsulfuration pathway through its primary reaction of catalyzing the formation of cysteine from cystathionine. The Neurospora crassa cystathionine γ-lyase gene (cys-16(+)) is of particular interest in dissecting the regulation and dynamics of transsulfuration. The aim of this study was to determine the regulatory connection of cys-16(+) to the Neurospora sulfur regulatory network. In addition, the cys-16(+) promoter was characterized with the goal of developing a strongly expressed and regulatable gene expression tool.
  
  
  RESULTS
  The cystathionine γ-lyase cys-16(+) gene was cloned and characterized. The gene, which contains no introns, encodes a protein of 417 amino acids with conserved pyridoxal 5'-phosphate binding site and substrate-cofactor binding pocket. Northern blot analysis using wild type cells showed that cys-16(+) transcript levels increased under sulfur limiting (derepressing) conditions and were present only at a low level under sulfur sufficient (repressing) conditions. In contrast, cys-16(+) transcript levels in a Δcys-3 regulatory mutant were present at a low level under either derepressing or repressing conditions. Gel mobility shift analysis demonstrated the presence of four CYS3 transcriptional activator binding sites on the cys-16(+) promoter, which were close matches to the CYS3 consensus binding sequence.
  
  
  CONCLUSIONS
  In this work, we confirm the control of cystathionine γ-lyase gene expression by the CYS3 transcriptional activator through the loss of cys-16(+) expression in a Δcys-3 mutant and through the in vitro binding of CYS3 to the cys-16(+) promoter at four sites. The highly regulated cys-16(+) promoter should be a useful tool for gene expression studies in Neurospora.
authors: Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
authors: Brad S
authors: BS
authors: Reveal
authors: John V
authors: JV
authors: Paietta
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003540:Cystathionine / Q000378:metabolism; D003542:Cystathionine gamma-Lyase / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000096:biosynthesis; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D053263:Gene Regulatory Networks; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D003540:Cystathionine; D011732:Pyridoxal Phosphate; D013455:Sulfur; D003542:Cystathionine gamma-Lyase; D003545:Cysteine