Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.1 (nitrate reductase)
3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zinc-finger domain/beta-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.
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PMID:NIT2, the nitrogen regulatory protein of Neurospora crassa, binds upstream of nia, the tomato nitrate reductase gene, in vitro. 153 Nov 84

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.
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PMID:Site-directed mutagenesis of the 'zinc finger' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora. 215 May 39

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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PMID: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. 759 72

The expression of the structural genes nit-3 and nit-6, which encode the nitrate assimilatory enzymes nitrate reductase and nitrite reductase, respectively, is highly regulated by the global-acting NIT2 regulatory protein. These structural genes are also controlled by nitrogen catabolite repression and by specific induction via nitrate. A pathway-specific regulatory protein, NIT4, appears to mediate nitrate induction of nit-3 and of nit-6. The NIT4 protein, composed of 1090 amino acids, contains a putative GAL4-like Cys-6 zinc cluster DNA-binding motif, which is joined by a short segment to a stretch of amino acids that appear to constitute a coiled-coil dimerization domain. Chemical crosslinking studies demonstrated that a truncated form of NIT4 forms homodimers. Mobility-shift and DNA-footprinting experiments have identified two NIT4-binding sites of significantly different strengths in the promoter region of the nit-3 gene. The stronger binding site contains a symmetrical octameric sequence, TCCGCGGA, whereas the weaker site has a related sequence. Sequences related to this palindromic element can be found upstream of the nit-6 gene.
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PMID:Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora. 759 94

The nit-3 gene of the filamentous fungus Neurospora crassa encodes nitrate reductase, the enzyme which catalyzes the first step in nitrate assimilation. The nit-3 gene is subject to a high degree of regulation by metabolic inducers and repressors, and its expression requires two distinct trans-acting regulatory proteins. Hypersensitive sites in the 5' DNA sequence upstream of the nit-3 gene were mapped with the use of three different nucleases as molecular probes. Six hypersensitive sites, three of which are very strong, were detected at essentially identical positions by all three nucleases. The hypersensitive sites appear to develop in a constitutive fashion and are present under conditions in which the nit-3 structural gene is expressed but also when this gene is inactive, although these sites are considerably less prominent in cells subjected to nitrogen catabolite repression. The presence of the hypersensitive sites appears to depend upon both the positively acting NIT2 and the positively acting NIT4 regulatory proteins, which might play a role in positioning of chromatin protein.
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PMID:Hypersensitive sites in the 5' promoter region of nit-3, a highly regulated structural gene of Neurospora crassa. 822 16

The NIT2 nitrogen regulatory protein of Neurospora is a DNA binding protein which contains a single Cys2/Cys2 type finger motif followed immediately by a highly basic region. Several different approaches were employed to identify nucleotides which appear to be in contact with NIT2 in the DNA-protein complex. Methylation interference and missing contact analyses with the promoter DNA fragment of the L-amino acid oxidase gene showed that all three purines in both of two GATA core sequences and the single adenine residue in each of the complementary TATC sequences were in intimate contact with NIT2. Modification or loss of the three purine residues located between the two GATA core sequences also significantly reduced NIT2 binding, whereas alteration of purines which flank the binding element showed only minor effects. Chemical modification of all six thymine bases in the two GATA and TATC complement core sequences also strongly affected NIT2 binding. High affinity NIT2 binding sites appear to contain at least two GATA core sequences, with single GATA sequences acting only as weak binding sites. Mobility shift experiments with the DNA fragment upstream of nit-3, the structural gene for nitrate reductase, revealed two DNA-NIT2 protein complexes. In complex I, which is formed first, NIT2 was bound to a pair of GATA sites located at -180. In complex II, the paired GATA sites at -180 plus a single GATA site at -290 were all occupied by NIT2. A DNA fragment containing only the single -290 GATA element bound NIT2 very weakly. The affinity of this single GATA for NIT2 was ten to twenty times greater when it was situated on the same DNA fragment with the distant paired GATA elements than when alone.
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PMID:Recognition of specific nucleotide bases and cooperative DNA binding by the trans-acting nitrogen regulatory protein NIT2 of Neurospora crassa. 839 61

Expression of nit-3 and nit-6, the structural genes which encode nitrate reductase and nitrite reductase in Neurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys6/Zn2 zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo in N. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.
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PMID:The regulatory protein NIT4 that mediates nitrate induction in Neurospora crassa contains a complex tripartite activation domain with a novel leucine-rich, acidic motif. 866 93

The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.
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PMID:Characterization of the Aspergillus parasiticus niaD and niiA gene cluster. 866 12

Nitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys2/Cys2 zinc finger DNA-binding domain. The negative-acting NMR protein interacts via specific protein-protein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2-NMR protein-protein interactions. These results provide compelling evidence that the specific NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.
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PMID:Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa. 942 2

In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions.
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PMID:Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa. 963 83


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