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. 1995 Nov;177(21):6093–6099. doi: 10.1128/jb.177.21.6093-6099.1995

Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa.

T Y Chiang 1, G A Marzluf 1
PMCID: PMC177447  PMID: 7592372

Abstract

In the filamentous fungus Neurospora crassa, both the global-acting regulatory protein NIT2 and the pathway-specific regulatory protein NIT4 are required to turn on the expression of the nit-3 gene, which encodes nitrate reductase, the first enzyme in the nitrate assimilatory pathway. Three NIT2 binding sites and two NIT4 binding sites have been identified in the 1.3-kb nit-3 promoter region via mobility shift and footprinting experiments with NIT2-beta-galactosidase and NIT4-beta-Gactosidase fusion proteins. Quantitative mobility shift assays were used to examine the affinity of individual NIT2 binding sites for the native NIT2 protein present in N. crassa nuclear extracts. In vivo analysis of nit-3 promoter 5' deletion constructs and individual NIT2 and NIT4 binding-site deletions or mutations revealed that all of the NIT2 and NIT4 binding sites are required for the full level of expression of the nit-3 gene. A cluster of two NIT2 and two NIT4 binding sites located more than 1 kb upstream of the translational start site is required for nit-3 expression, and one NIT2 binding site and one NIT4 site, which are immediately adjacent to each other, are of particular functional importance. A significant NIT2-NIT4 protein-protein interaction might occur upon their binding to nearby sites.

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Selected References

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  1. Chiang T. Y., Marzluf G. A. DNA recognition by the NIT2 nitrogen regulatory protein: importance of the number, spacing, and orientation of GATA core elements and their flanking sequences upon NIT2 binding. Biochemistry. 1994 Jan 18;33(2):576–582. doi: 10.1021/bi00168a024. [DOI] [PubMed] [Google Scholar]
  2. Chiang T. Y., Rai R., Cooper T. G., Marzluf G. A. DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: analysis with mutated binding sites. Mol Gen Genet. 1994 Nov 15;245(4):512–516. doi: 10.1007/BF00302264. [DOI] [PubMed] [Google Scholar]
  3. Exley G. E., Colandene J. D., Garrett R. H. Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa. J Bacteriol. 1993 Apr;175(8):2379–2392. doi: 10.1128/jb.175.8.2379-2392.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fu Y. H., Feng B., Evans S., Marzluf G. A. Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora. Mol Microbiol. 1995 Mar;15(5):935–942. doi: 10.1111/j.1365-2958.1995.tb02362.x. [DOI] [PubMed] [Google Scholar]
  5. Fu Y. H., Marzluf G. A. Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa. Mol Cell Biol. 1987 May;7(5):1691–1696. doi: 10.1128/mcb.7.5.1691. [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., Marzluf G. A. nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol. 1990 Mar;10(3):1056–1065. doi: 10.1128/mcb.10.3.1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fu Y. H., Marzluf G. A. nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5331–5335. doi: 10.1073/pnas.87.14.5331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garrett R. H., Cove D. J. Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants. Mol Gen Genet. 1976 Dec 8;149(2):179–186. doi: 10.1007/BF00332887. [DOI] [PubMed] [Google Scholar]
  10. Hautala J. A., Conner B. H., Jacobson J. W., Patel G. L., Giles N. H. Isolation and characterization of nuclei from Neurospora crassa. J Bacteriol. 1977 May;130(2):704–713. doi: 10.1128/jb.130.2.704-713.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnstone I. L., McCabe P. C., Greaves P., Gurr S. J., Cole G. E., Brow M. A., Unkles S. E., Clutterbuck A. J., Kinghorn J. R., Innis M. A. Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene. 1990 Jun 15;90(2):181–192. doi: 10.1016/0378-1119(90)90178-t. [DOI] [PubMed] [Google Scholar]
  12. Lee H., Fu Y. H., Marzluf G. A. Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa. Biochemistry. 1990 Sep 18;29(37):8779–8787. doi: 10.1021/bi00489a039. [DOI] [PubMed] [Google Scholar]
  13. Marzluf G. A. Regulation of nitrogen metabolism and gene expression in fungi. Microbiol Rev. 1981 Sep;45(3):437–461. doi: 10.1128/mr.45.3.437-461.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marzluf G. A. Regulation of sulfur and nitrogen metabolism in filamentous fungi. Annu Rev Microbiol. 1993;47:31–55. doi: 10.1146/annurev.mi.47.100193.000335. [DOI] [PubMed] [Google Scholar]
  15. Okamoto P. M., Garrett R. H., Marzluf G. A. Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa. Mol Gen Genet. 1993 Apr;238(1-2):81–90. doi: 10.1007/BF00279534. [DOI] [PubMed] [Google Scholar]
  16. Punt P. J., Strauss J., Smit R., Kinghorn J. R., van den Hondel C. A., Scazzocchio C. The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally. Mol Cell Biol. 1995 Oct;15(10):5688–5699. doi: 10.1128/mcb.15.10.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Selker E. U., Garrett P. W. DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6870–6874. doi: 10.1073/pnas.85.18.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Xiao X., Fu Y. H., Marzluf G. A. The negative-acting NMR regulatory protein of Neurospora crassa binds to and inhibits the DNA-binding activity of the positive-acting nitrogen regulatory protein NIT2. Biochemistry. 1995 Jul 11;34(27):8861–8868. doi: 10.1021/bi00027a038. [DOI] [PubMed] [Google Scholar]
  19. Yuan G. F., Fu Y. H., Marzluf G. A. nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol Cell Biol. 1991 Nov;11(11):5735–5745. doi: 10.1128/mcb.11.11.5735. [DOI] [PMC free article] [PubMed] [Google Scholar]

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