Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa.
The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.
Paietta
J V
JV
Department of Biochemistry, Wright State University, Dayton, Ohio 45435.
D001192:Arylsulfatases / Q000235:genetics; D001483:Base Sequence; D003542:Cystathionine gamma-Lyase; D004268:DNA-Binding Proteins; D004794:Enzyme Repression / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005809:Genes, Regulator / Q000235:genetics*; D005816:Genetic Complementation Test; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013455:Sulfur / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014404:Tubulin; D013455:Sulfur; D001192:Arylsulfatases; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase