Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Nov;11(11):5735–5745. doi: 10.1128/mcb.11.11.5735

nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

G F Yuan 1, Y H Fu 1, G A Marzluf 1
PMCID: PMC361945  PMID: 1840634

Abstract

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

Full text

PDF
5736

Selected References

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

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baum J. A., Geever R., Giles N. H. Expression of qa-1F activator protein: identification of upstream binding sites in the qa gene cluster and localization of the DNA-binding domain. Mol Cell Biol. 1987 Mar;7(3):1256–1266. doi: 10.1128/mcb.7.3.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beri R. K., Whittington H., Roberts C. F., Hawkins A. R. Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans. Nucleic Acids Res. 1987 Oct 12;15(19):7991–8001. doi: 10.1093/nar/15.19.7991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bricmont P. A., Daugherty J. R., Cooper T. G. The DAL81 gene product is required for induced expression of two differently regulated nitrogen catabolic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Feb;11(2):1161–1166. doi: 10.1128/mcb.11.2.1161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burger G., Tilburn J., Scazzocchio C. Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans. Mol Cell Biol. 1991 Feb;11(2):795–802. doi: 10.1128/mcb.11.2.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chasman D. I., Kornberg R. D. GAL4 protein: purification, association with GAL80 protein, and conserved domain structure. Mol Cell Biol. 1990 Jun;10(6):2916–2923. doi: 10.1128/mcb.10.6.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Corton J. C., Johnston S. A. Altering DNA-binding specificity of GAL4 requires sequences adjacent to the zinc finger. Nature. 1989 Aug 31;340(6236):724–727. doi: 10.1038/340724a0. [DOI] [PubMed] [Google Scholar]
  9. Courey A. J., Holtzman D. A., Jackson S. P., Tjian R. Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell. 1989 Dec 1;59(5):827–836. doi: 10.1016/0092-8674(89)90606-5. [DOI] [PubMed] [Google Scholar]
  10. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  11. Dunn-Coleman N. S., Tomsett A. B., Garrett R. H. The regulation of nitrate assimilation in Neurospora crassa: biochemical analysis of the nmr-1 mutants. Mol Gen Genet. 1981;182(2):234–239. doi: 10.1007/BF00269663. [DOI] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. Friden P., Schimmel P. LEU3 of Saccharomyces cerevisiae encodes a factor for control of RNA levels of a group of leucine-specific genes. Mol Cell Biol. 1987 Aug;7(8):2708–2717. doi: 10.1128/mcb.7.8.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedman D. I., Imperiale M. J., Adhya S. L. RNA 3' end formation in the control of gene expression. Annu Rev Genet. 1987;21:453–488. doi: 10.1146/annurev.ge.21.120187.002321. [DOI] [PubMed] [Google Scholar]
  15. Fu Y. H., Kneesi J. Y., Marzluf G. A. Isolation of nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in Neurospora crassa. J Bacteriol. 1989 Jul;171(7):4067–4070. doi: 10.1128/jb.171.7.4067-4070.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Fu Y. H., Marzluf G. A. Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8243–8247. doi: 10.1073/pnas.84.23.8243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Fu Y. H., Young J. L., Marzluf G. A. Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa. Mol Gen Genet. 1988 Sep;214(1):74–79. doi: 10.1007/BF00340182. [DOI] [PubMed] [Google Scholar]
  22. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  23. Hawker K. L., Montague P., Marzluf G. A., Kinghorn J. R. Heterologous expression and regulation of the Neurospora crassa nit-4 pathway-specific regulatory gene for nitrate assimilation in Aspergillus nidulans. Gene. 1991 Apr;100:237–240. doi: 10.1016/0378-1119(91)90373-j. [DOI] [PubMed] [Google Scholar]
  24. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  25. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kammerer B., Guyonvarch A., Hubert J. C. Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage. J Mol Biol. 1984 Dec 5;180(2):239–250. doi: 10.1016/s0022-2836(84)80002-9. [DOI] [PubMed] [Google Scholar]
  27. Kao C. C., Lieberman P. M., Schmidt M. C., Zhou Q., Pei R., Berk A. J. Cloning of a transcriptionally active human TATA binding factor. Science. 1990 Jun 29;248(4963):1646–1650. doi: 10.1126/science.2194289. [DOI] [PubMed] [Google Scholar]
  28. Kim J., Michels C. A. The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein. Curr Genet. 1988 Oct;14(4):319–323. doi: 10.1007/BF00419988. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Messenguy F., Dubois E., Descamps F. Nucleotide sequence of the ARGRII regulatory gene and amino acid sequence homologies between ARGRII PPRI and GAL4 regulatory proteins. Eur J Biochem. 1986 May 15;157(1):77–81. doi: 10.1111/j.1432-1033.1986.tb09640.x. [DOI] [PubMed] [Google Scholar]
  33. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  34. Orbach M. J., Porro E. B., Yanofsky C. Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol. 1986 Jul;6(7):2452–2461. doi: 10.1128/mcb.6.7.2452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pan T., Coleman J. E. GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2077–2081. doi: 10.1073/pnas.87.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pfeifer K., Kim K. S., Kogan S., Guarente L. Functional dissection and sequence of yeast HAP1 activator. Cell. 1989 Jan 27;56(2):291–301. doi: 10.1016/0092-8674(89)90903-3. [DOI] [PubMed] [Google Scholar]
  37. Reinert W. R., Patel V. B., Giles N. H. Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency on qa-1 function. Mol Cell Biol. 1981 Sep;1(9):829–835. doi: 10.1128/mcb.1.9.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schultz J., Marshall-Carlson L., Carlson M. The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4744–4756. doi: 10.1128/mcb.10.9.4744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sorger G. J., Giles N. H. Genetic control of nitrate reductase in Neurospora crassa. Genetics. 1965 Oct;52(4):777–788. doi: 10.1093/genetics/52.4.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tomsett A. B., Garrett R. H. Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase. Mol Gen Genet. 1981;184(2):183–190. doi: 10.1007/BF00272903. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Wharton K. A., Yedvobnick B., Finnerty V. G., Artavanis-Tsakonas S. opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster. Cell. 1985 Jan;40(1):55–62. doi: 10.1016/0092-8674(85)90308-3. [DOI] [PubMed] [Google Scholar]
  44. Witte M. M., Dickson R. C. Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis. Mol Cell Biol. 1988 Sep;8(9):3726–3733. doi: 10.1128/mcb.8.9.3726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Witte M. M., Dickson R. C. The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1. Mol Cell Biol. 1990 Oct;10(10):5128–5137. doi: 10.1128/mcb.10.10.5128. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES