Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization, autogenous control, and regions required for transcriptional activation

  • 62 Accesses

  • 20 Citations

Abstract

The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism. CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain. Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated. The CYS3 protein was not observed in cys-3 mutants. Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli. A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein. Amino-terminal, carboxy-terminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function. Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity. All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function. Alanine scanning mutagenesis demonstrated that an unusual proline-rich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain. It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.

This is a preview of subscription content, log in to check access.

References

  1. Burton EG, Metzenberg RL (1971) Novel mutation causing derepression of several enzymes of sulfur metabolism in Neurospora crassa. J Bacteriol 109:140–151

  2. Courey AJ, Tjian R (1988) Analysis of SP1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887–898

  3. Comai L, Tanese N, Tjian R (1992) The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell 68:965–976

  4. Desplan C, Theis J, O'Farrel PH (1988) The sequence specificity of homeodomain-DNA interaction. Cell 54:1081–1090

  5. Dynlacht BD, Hoey T, Tjian R (1991) Isolation of coactivators associated with TATA-binding protein that mediate transcriptional activation. Cell 66:563–576

  6. Ebbole D, Sachs MS (1990) A rapid -and simple method for isolation of Neurospora crassa homokaryons using microconidia. Neurospora Newslett 37:17–18

  7. Fu YH, Marzluf GA (1990) Cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. J Biol Chem 265:11942–11947

  8. Fu YH, Paietta JV, Mannix DG, Marzluf GA (1989) Cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element. Mol Cell Biol 9:1120–1127

  9. Fu YH, Lee HJ, Young JL, Jarai G, Marzluf GA (1990) Nitrogen and sulfur regulatory circuits of Neurospora. In: Davidson EH, Ruderman JV, Posakony JW (ds) Developmental Biology. Wiley-Liss, pp 319–335

  10. Godowski PJ, Picard D, Yamamoto KR (1988) Signal transduction and transcriptional regulation by glucocorticoid receptor-lexA fusion proteins. Science 241:812–816

  11. Hautala JA, Conner BH, Jacobson JW, Patel GL, Giles NH (1977) Isolation and characterization of nucleic from Neurospora crassa. J Bacteriol 130:704–713

  12. Hope IA, Struhl K (1986) Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46:885–894

  13. Hope IA, Mahadevan S, Struhl K (1988) Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein. Nature 333:635–640

  14. Kanaan MN, Marzluf GA (1991) Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa. Mol Cell Biol 9:4356–4362

  15. Kanaan MN, Fu YH, Marzluf GA (1992) The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite. Biochemistry 31:3197–3203

  16. Ketter JS, Marzluf GA (1988) Molecular cloning and analysis of the regulation of cys-14, a structural gene of the sulfur regulatory circuit of Neurospora crassa. Mol Cell Biol 8:1504–1508

  17. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492

  18. Ma J, Ptashne M (1987) Deletion analysis of GAL4 defines two transcriptional activating segments. Cell 48:847–853

  19. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  20. Marzluf GA, Metzenberg RL (1968) Positive control by the cys-3 locus in regulation of sulfur metabolism in Neurospora crassa. J Mol Biol 33:423–437

  21. Mermond N, O'Neill EA, Kelly TJ, Tjian R (1989) The proline-rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA-binding domain. Cell 58:741–753

  22. Metzenberg RL, Parson JW (1966) Altered repression of some enzymes of sulfur utilization in a temperature-conditional lethal mutant of Neurospora. Proc Natl Acad Sci USA 55:629–635

  23. Paietta JV (1989) Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa. Mol Cell Biol 9:3630–3637

  24. Paietta JV (1990) Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa. Mol Cell Biol 10:5207–5214

  25. Paietta JV (1992) Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa. Mol Cell Biol 12:1568–1577

  26. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

Download references

Author information

Correspondence to George A. Marzluf.

Additional information

Communicated by C. van den Hondel

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kanaan, M.N., Marzluf, G.A. The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization, autogenous control, and regions required for transcriptional activation. Molec. Gen. Genet. 239, 334–344 (1993). https://doi.org/10.1007/BF00276931

Download citation

Key words

  • Neurospora crassa
  • CYS3 protein
  • Sulfur regulation
  • Autogenous control
  • Trans-activation