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. 1991 Sep;11(9):4356–4362. doi: 10.1128/mcb.11.9.4356

Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

M N Kanaan 1, G A Marzluf 1
PMCID: PMC361297  PMID: 1831537

Abstract

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agre P., Johnson P. F., McKnight S. L. Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP. Science. 1989 Nov 17;246(4932):922–926. doi: 10.1126/science.2530632. [DOI] [PubMed] [Google Scholar]
  2. Bohmann D., Tjian R. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell. 1989 Nov 17;59(4):709–717. doi: 10.1016/0092-8674(89)90017-2. [DOI] [PubMed] [Google Scholar]
  3. Burton E. G., Metzenberg R. L. Novel mutation causing derepression of several enzymes of sulfur metabolism in Neurospora crassa. J Bacteriol. 1972 Jan;109(1):140–151. doi: 10.1128/jb.109.1.140-151.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  5. Fu Y. H., Marzluf G. A. Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa. J Bacteriol. 1988 Feb;170(2):657–661. doi: 10.1128/jb.170.2.657-661.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fu Y. H., Marzluf G. A. cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. J Biol Chem. 1990 Jul 15;265(20):11942–11947. [PubMed] [Google Scholar]
  7. Fu Y. H., Paietta J. V., Mannix D. G., Marzluf G. A. 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. 1989 Mar;9(3):1120–1127. doi: 10.1128/mcb.9.3.1120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gentz R., Rauscher F. J., 3rd, Abate C., Curran T. Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains. Science. 1989 Mar 31;243(4899):1695–1699. doi: 10.1126/science.2494702. [DOI] [PubMed] [Google Scholar]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
  11. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  12. Marzluf G. A., Metzenberg R. L. Positive control by the cys-3 locus in regulation of sulfur metabolism in Neurospora. J Mol Biol. 1968 Apr 28;33(2):423–437. doi: 10.1016/0022-2836(68)90199-x. [DOI] [PubMed] [Google Scholar]
  13. O'Neil K. T., Hoess R. H., DeGrado W. F. Design of DNA-binding peptides based on the leucine zipper motif. Science. 1990 Aug 17;249(4970):774–778. doi: 10.1126/science.2389143. [DOI] [PubMed] [Google Scholar]
  14. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  15. Paietta J. V., Akins R. A., Lambowitz A. M., Marzluf G. A. Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa. Mol Cell Biol. 1987 Jul;7(7):2506–2511. doi: 10.1128/mcb.7.7.2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shuman J. D., Vinson C. R., McKnight S. L. Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP. Science. 1990 Aug 17;249(4970):771–774. doi: 10.1126/science.2202050. [DOI] [PubMed] [Google Scholar]
  17. Talanian R. V., McKnight C. J., Kim P. S. Sequence-specific DNA binding by a short peptide dimer. Science. 1990 Aug 17;249(4970):769–771. doi: 10.1126/science.2389142. [DOI] [PubMed] [Google Scholar]
  18. Turner R., Tjian R. Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science. 1989 Mar 31;243(4899):1689–1694. doi: 10.1126/science.2494701. [DOI] [PubMed] [Google Scholar]
  19. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  20. Vollmer S. J., Yanofsky C. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]

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