Abstract
NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5′ or 3′ sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.
This is a preview of subscription content, log in via an institution to check access.
References
Bysani N, Daugherty JR, Cooper TC (1991) Saturation mutagenesis of the UASNTR (GATAA) responsible for nitrogen catabolite repression-sensitive transcriptional activation of the allantoin pathway genes in Saccharomyces cerevisiae. J Bacteriol 173:4977–4982
Caddick MX, Arst HN, Taylor LH, Johnson RI, Brownlee AG (1986) Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. EMBO J 5:1087–1090
Cunningham TS, Cooper TG (1993) The Saccharomyces cerevisiae DAL80 repressor protein binds to multiple copies of GATAA-containing sequences (URSGATA). J Bacteriol 175:5851–5861
Daugherty JR, Rai R, ElBerry HM, Cooper TG (1993) Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharmomyces cerevisiae. J Bacteriol 175:64–73
Eisen A, Taylor WE, Blumberg H, Young ET (1988) The yeast regulatory protein ADRI binds in a zinc-dependent manner to the upstream activation sequence of ADH2. Mol Cell Biol 8:4552–4556
Evans T, Felsenfeld G (1989) The erythroid-specific transcription factor Eryfl: a new finger protein. Cell 58:877–885
Fu YH, Marzluf GA (1987) Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa. Mol Cell Biol 7:1691–1696
Fu YH, Marzluf GA (1988) Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa. J Bacteriol 170:657–661
Fu YH, Marzluf GA (1990a) nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol 10:1056–1065
Fu YH, Marzluf GA (1990b) nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequencespecific DNA-binding protein. Proc Natl Acad Sci USA 87:5331–5335
Ko LJ, Engel JD (1993) DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol 13:4011–4022
Lee HJ, Fu YH, Marzluf GA (1990) Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa. Biochemistry 29:8779–8787
Marzluf GA (1981) Regulation of nitrogen metabolism and gene expression in fungi. Microbiol Rev 45:437–461
Marzluf GA (1993) Regulation of sulfur and nitrogen metabolism in filamentous fungi. Ann Rev Microbiol 47:31–55
Merika M, Orkin SH (1993) DNA-binding specificity of GATA family transcription factors. Mol Cell Biol 13:3999–4010
Metzenberg RL (1979) Implications of some genetic control mechanisms in Neurospora. Microbiol Rev 43:361–383
Minehart PL, Magasanik B (1991) Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-Dinning domain. Mol Cell Biol 12:6216–6226
Omichinski JG, Clore GM, Schaad O, et al (1993) NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. Science 261:438–446
Orkin SH (1992) GATA-binding transcription factors in hematopoietic cells. Blood 80:575–581
Rai R, Genbauffe FS, Sumrada RA, Cooper TC (1989) Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae. Mol Cell Biol 9:602–608
Spieth J, Shim YH, Lea K, Conrad R, Blumenthal T (1991) elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family. Mol Cell Biol 11:4651–4659
Xiao XD, Marzluf GA (1993) Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa, alter promoter element recognition. Curr Genet 24:212–218
Zon LI, Tsai SF, Burgess S, Matsudaira P, Bruns GA, Orkin SH (1990) The major human erythroid DNA-binding protein (GF-1): Primary sequence and localization of the gene to the X chromosome. Proc Natl Acad Sci USA 87:668–672
Author information
Authors and Affiliations
Additional information
Communicated by C. van den Hondel
Rights and permissions
About this article
Cite this article
Chiang, TY., Rai, R., Cooper, T.G. et al. DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: Analysis with mutated binding sites. Molec. Gen. Genet. 245, 512–516 (1994). https://doi.org/10.1007/BF00302264
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00302264