NIT2 Protein Research Articles

Crystal Structure of Nitrilase-Like Protein Nit2 from Kluyveromyces lactis

The nitrilase superfamily, including 13 branches, plays various biological functions in signaling molecule synthesis, vitamin metabolism, small-molecule detoxification, and posttranslational modifications. Most of the mammals and yeasts have Nit1 and Nit2 proteins, which belong to the nitrilase-like (Nit) branch of the nitrilase superfamily. Recent studies have suggested that Nit1 is a metabolite repair enzyme, whereas Nit2 shows ω-amidase activity. In addition, Nit1 and Nit2 are suggested as putative tumor suppressors through different ways in mammals. Yeast Nit2 (yNit2) is a homolog of mouse Nit1 based on similarity in sequence. To understand its specific structural features, we determined the crystal structure of Nit2 from Kluyveromyces lactis (KlNit2) at 2.2 Å resolution and compared it with the structure of yeast-, worm-, and mouse-derived Nit2 proteins. Based on our structural analysis, we identified five distinguishable structural features from 28 structural homologs. This study might potentially provide insights into the structural relationships of a broad spectrum of nitrilases.

Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity.

The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inabilityto grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of itsincreased transcription levels of nit1 and nit2genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1∆nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of the fungus. These observations open new questions about the role of Nit1, Nit2, and Nit3 proteins in other routes of nitrate metabolism in this pathogenic fungus and in the possible regulatory role that can be exerted by the AreA protein in these routes.

Chlamydomonas NZF1, a tandem‐repeated zinc finger factor involved in nitrate signalling by controlling the regulatory gene NIT2

The Chlamydomonas reinhardtii NIT2 gene plays a central role in nitrate assimilation, thus, nit2 mutants are not able to sense or to use nitrate for growth. NIT2 protein is an RWP-RK-type transcriptional factor related to nodule inception (Nin, NLP) proteins from plants. NIT2 expression is down-regulated in ammonium and up-regulated under nitrogen deprivation. However, intracellular nitrate is required to activate NIT2 for subsequent expression of NIA1 and other nitrate assimilation genes. In this work, mutants defective in nitrate sensing have been studied. The identification of genomic regions affected allows proposing putative loci/genes for nitrate signalling in the alga. Among them, a CrNZF1 (Nitrate Zinc Finger 1) that encodes a tandem zinc finger protein CCCH-type. In the nzf1 mutant, the expression of the regulatory gene NIT2 is decreased and also that of nitrate assimilation genes. In this mutant, polyadenylated forms of NIT2 with different lengths could be detected, whereas in the wild type there appeared preferentially the longest forms. CrNZF1 is proposed to regulate NIT2 polyadenylation and thus nitrate signalling and nitrate-dependent growth in the alga.

A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen Metabolism

Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression.

Nitrate Signaling by the Regulatory GeneNIT2inChlamydomonas

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit(-) (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.

Characterization of pco-1, a newly identified gene which regulates purine catabolism in Neurospora.

A new gene of Neurospora crassa, designated pco-1, was characterized and shown to regulate the expression of several genes which encode enzymes required for the catabolism of purines. Unlike the wild type, a pco-1 mutant created by repeat-induced point mutation cannot utilize purines as a nitrogen source. The PCO1 protein contains a Zn(II)2Cys6 binuclear cluster motif near its N-terminus, followed by a putative coiled-coil motif. A chemical crosslinking experiment demonstrated that PCO1 forms homodimers. PCO1 binds to CGG-N6-CCG elements located in the upstream promoter region of four genes encoding purine catabolic enzymes. Northern blot analysis demonstrated that a functional PCO1 protein is required for induction of xdh, which encodes xanthine dehydrogenase. Moreover, PCO1 was required for induction of three different purine catabolic enzymes. Two glutamine-rich domains occur in the C-terminal region of PCO1 and at least one of the glutamine-rich regions is required for PCO1 function, suggesting that they might play a role in transcriptional activation. The PCO1 protein does not interact with the global-acting NIT2 protein or the negative-acting NMR protein that functions in nitrogen catabolite repression. Induction of the xdh gene and synthesis of xanthine dehydrogenase is completely dependent upon PCO1, but does not require the global-acting NIT2 protein, suggesting that it is controlled by a novel regulatory mechanism.

Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression.

Nitrate reductase (NR), the rate-limiting and primary control point of the nitrate assimilation pathway, is regulated at transcriptional and post-transcriptional levels. To better understand how NR is regulated at the transcriptional level in Chlorella vulgaris, studies were performed to identify the factors regulating NR expression. Sequence analysis of the NR promoter identified a number of potential sites that were investigated by mobility shift assays. Of the protein-binding sites found, several., such as USF and E2F are likely involved in the basal NR gene transcription. An indirect repeat sequence with similarity to the sequence recognized by the common plant regulatory factor was identified and shown to bind a Chlorella protein in vitro. Mobility shift assays of a consensus GATA element indicated that proteins able to specifically bind this element are constitutive, regardless of the nitrogen source. Mutational analysis revealed that the GATA core is required for protein binding in vitro. Additionally, a NIT2 zinc-finger domain/glutathione S-transferase fusion protein was found to bind in a sequence-specific manner to this site. In Neurospora crassa and Aspergillus nidulans, consensus GATA elements are bound by the NIT2 protein, which has a major role in the expression of nitrogen-metabolizing genes. The ability of the GATA element to function as a nitrogen response element (NRE) was further examined by in vivo foot-printing. The protection of guanines in the GATA core and surrounding region was observed only in cells grown in the presence of nitrate. These data confirm that a single GATA element has a role in regulating the expression of NR in C. vulgaris.

Analysis of a distal cluster of binding elements and other unusual features of the promoter of the highly regulated nit-3 gene of Neurospora crassa.

In Neurospora crassa, the expression of the nit-3 gene (nitrate reductase) is dependent upon nitrogen derepression and nitrate induction and is regulated by two positive-acting transcription factors, NIT2 and NIT4, and a negative regulator, NMR. The presence of a tightly linked cluster of NIT2 and NIT4 binding sites suggested that their close spacing might be required for a synergistic interaction of the NIT2 and NIT4 proteins. We show here that the NIT2 and NIT4 binding sites can be separated without affecting either the expression level or the precise regulation of the nit-3 gene. Studies conducted on the NIT2 site II, which contains only a single GATA element and yet plays a major role in nit-3 gene expression, showed that nucleotides both 5' and 3' of the GATA sequence were important for strong DNA binding in vitro and its activation function in vivo. The nit-3 promoter contains two long AT-rich sequences, one of which is located just upstream of the transcription start sites and is required for optimal promoter function. The nit-3 transcript contains eight TACC repeats in its 5' noncoding region which appear to be involved in mRNA instability. Deletion of these TACC repeats led to a significant increase in the stability of nit-3 mRNA.

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.

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.

Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa.

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.

Genetic regulation of nitrogen metabolism in the fungi.

In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

Genetic regulation of nitrogen metabolism in the fungi.

In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

Identification of the native NIT2 major nitrogen regulatory protein in nuclear extracts of Neurospora crassa.

The nit-2 gene of Neurospora crassa encodes the major nitrogen regulatory protein which acts in a positive fashion to activate the expression of many different structural genes during conditions of nitrogen limitation. An E. coli-expressed NIT2/beta-Gal fusion protein binds specifically to DNA in vitro by recognizing GATA core elements. Nuclear extracts prepared from a wild-type N. crassa strain contain a protein factor which displays all of the properties expected for the native NIT2 protein. The native NIT2 protein in nuclear extracts binds with high affinity to DNA fragments which contain two GATA elements, weakly to fragments with a single GATA element, and fails to bind to DNAs which lack these sequences. The DNA binding ability of the protein factor in nuclear extracts is efficiently blocked by a polyclonal antibody developed against the zinc-finger region of NIT2 protein. Western blot analysis with the anti-NIT2 antiserum revealed a specific protein with a size of approximately 110,000 daltons, in excellent agreement with the predicted size of NIT2. Both the specific NIT2 DNA binding activity and the protein detected by Western blot are totally lacking in nuclear extracts of a nit-2 rip mutant strain. These results all support the conclusion that the native NIT2 protein in Neurospora cells has been identified. The NIT2 protein is localised in nuclei and could not be detected in the cytoplasmic fraction of cells subjected to nitrogen derepression or nitrogen repression, indicating that the nuclear import of NIT2 is not regulated.

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

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.

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.

Structural genes of the nitrogen regulatory circuit of the filamentous fungus Neurospora crassa are under the control of both positive and negative regulatory proteins. NIT2, the major positive-acting nitrogen regulatory protein, activates the expression of structural genes within the nitrogen circuit. NIT2 binds to upstream activation sites which contain at least two GATA core elements in the promoter regions of the nitrogen-controlled structural genes, and activates their transcription, possibly by way of acidic activation domains. The mechanism by which a putative negative-acting regulator, NMR, mediates nitrogen repression of the various structural genes has remained unclear. In the studies reported here, a direct interaction between the NIT2 and NMR proteins has been demonstrated by the use of two different experimental approaches. The yeast two-hybrid system was used to show NIT2-NMR-specific binding in vivo; an independent in vitro assay for protein-protein binding also demonstrated a specific interaction between NIT2 and NMR. Two separate regions of the NIT2 protein, both of which appear to exist as alpha-helices, make direct contact with the NMR protein. One of these alpha-helical regions occurs within the zinc finger DNA-binding domain of NIT2. Mutant NIT2 proteins with amino acid substitutions in the zinc finger motif do not bind to NMR. Mobility shift experiments revealed that the NMR protein inhibits NIT2 DNA binding in vitro. We suggest that NMR carries out its negative regulatory role by directly binding to NIT2, and thereby blocking the function of NIT2 by inhibiting its DNA-binding activity.

DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: analysis with mutated binding sites.

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.

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter-beta-glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa, alter promoter element recognition.

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.

NIT2, the nitrogen regulatory protein of Neurospora crassa, binds upstream of nia, the tomato nitrate reductase gene, in vitro

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.

Sequence and Expression of GLN3, a Positive Nitrogen Regulatory Gene of Saccharomyces cerevisiae Encoding a Protein with a Putative Zinc Finger DNA-Binding Domain

The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability.