Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.     

cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein

cys-3, the positive-acting master sulfur regulatory gene of Neurospora crassa, turns on the expression of an entire set of unlinked structural genes which encode sulfur-catabolic enzymes. cys-3 encodes a protein of 236 amino acid residues and contains a potential bipartite DNA-binding domain which consists of a leucine zipper and an adjacent highly basic region. Gel band mobility shift and DNA footprint experiments were used to demonstrate that the CYS3 protein, expressed in Escherichia coli, binds to three distinct sites in the 5' upstream DNA of cys-14, the structural gene for sulfate permease II. The CYS3 protein also binds to one distinct sequence element upstream of the cys-3 gene itself, which suggests an autoregulatory role for this protein. Two mutant CYS3 proteins, altered in the basic region of the DNA-binding domain, failed to bind to either the cys-14 or the cys-3 upstream recognition elements.     

Novel Mutation Causing Derepression of Several Enzymes of Sulfur Metabolism in Neurospora crassa

A group of enzymes of sulfur metabolism (arylsulfatase, cholinesulfatase, and a number of others) are normally repressed in Neurospora crassa by an abundant supply of a "favored" sulfur source such as methionine or inorganic sulfate. A mutant called scon(c) was isolated in which the formation of each of these enzymes is largely or completely nonrepressible. The structural genes for three of these enzymes have been mapped; scon(c) is not linked to any of them. It is also not linked to cys-3, another gene which is involved in control of the same group of enzymes. Two alleles of the structural gene for arylsulfatase [ars(+) and ars(UFC-220)] produce electrophoretically distinguishable forms of arylsulfatase. Heterokaryons with the constitution scon(c) ars(+) + scon(+)ars(UFC-220) were prepared. These heterokaryons produce both forms of arylsulfatase under conditions of sulfur limitation, but produce only the wild-type (ars(+)) form under conditions of sulfur abundance. When the alleles of ars and scon are in the opposite relationship, only the ars(UFC-220) form of arylsulfatase can be detected under conditions of sulfur abundance. Thus the effect of the scon(c) mutation seems to be limited to its own nucleus. The implications of these findings are discussed.     

Metabolic suppressors of a regulatory mutant in Neurospora

The synthesis of a number of enzymes of sulfur assimilation in Neurospora crassa is controlled by the sulfur source. Mutants in one regulatory gene, cys-3, are unable to make any of the enzymes. This locus is thought to specify a macromolecule that is required for the expression of the structural genes. A mutant, scon ^c, of another regulatory gene is nonrepressible for the synthesis of the enzymes. We report here the isolation of suppressed scon ^c strains which are actually the double mutant, scon ^c,cys-3. These strains are phenotypically indistinguishable from single mutants at the cys-3 locus. Thus cys-3 is epistatic to scon ^c. Evidence that the expression of the cys-3 gene is itself controlled is also presented.This work is supported by a Public Health Service grant, GM-08995, from the National Institute of General Medical Sciences. One of us (R.L.M.) is supported by a Public Health Service Career Development Award.     

The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite.

Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

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.     

The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization,autogenous control,and regions required for transcriptional activation

The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism. CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain. Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated. The CYS3 protein was not observed in cys-3 mutants. Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli. A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein. Amino-terminal, carboxy-terminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function. Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity. All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function. Alanine scanning mutagenesis demonstrated that an unusual proline-rich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain. It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.     

Sulfate transport in Neurospora crassa: regulation, turnover, and cellular localization of the CYS-14 protein.

Uptake of inorganic sulfate in Neurospora crassa is governed by the sulfur regulatory circuit and is under the control of positively and negatively acting regulatory genes. Two genetically and biochemically distinct systems are responsible for the uptake of sulfate from the environment. One of these, sulfate permease II, encoded by the cys-14 gene, functions primarily in mycelia. A defined region of the CYS-14 protein was highly expressed in Escherichia coli and purified. Anti-CYS-14 antibody was produced and used to detect the CYS-14 protein in N. crassa extracts. The CYS-14 protein has an approximate molecular weight of 95K, in agreement with its calculated size based on its predicted amino acid sequence. The steady-state level of the CYS-14 protein is highly regulated in wild-type mycelia and constitutive in an scon-1 mutant, whereas no CYS-14 protein could be detected in a cys-3 mutant. Following the accumulation of the cys-14 mRNA, that reaches its maximum in about 6 h, the CYS-14 protein accumulates to a maximum level in about 8 h after derepression. During conditions of sulfur repression, the CYS-14 protein turns over with a half-life of approximately 2 h. The CYS-14 protein appears to be localized in the plasma membrane, suggesting that it functions as a sulfate ion transporter.     

Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.     

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.     

Analysis of CYS3 regulator function in Neurospora crassa by modification of leucine zipper dimerization specificity.

The CYS3 positive regulator is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of sulfur-controlled structural genes in Neurospora crassa. An approach of modifying the dimerization specificity of the CYS3 leucine zipper was used to determine whether the in vivo regulatory function of CYS3 requires the formation of homodimeric or heterodimeric complexes. Two altered versions of CYS3 with coiled coil elecrostatic interactions favorable to heterodimerization showed restoration of wild-type CYS3 function only when simultaneously expressed in a delta cys-3 strain. In addition, constructs having the CYS3 leucine zipper swapped for that of the oncoprotein Jun or the CYS3 leucine zipper extended by a heptad repeat showed wild-type CYS3 function when transformed into a delta cys-3 strain. Gel mobility shift and immunoprecipitation assays were used to confirm the modified CYS3 proteins dimerization and DNA binding properties. The studies, which precluded wild-type CYS3 dimerization, indicate that in vivo CYS3 is fully functional as a homodimer since no interaction was required with other leucine zipper proteins to activate sulfur regulatory and structural gene expression. The results demonstrate the utility of leucine zipper modification to study the in vivo function of bZIP proteins.     

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Nucleotide sequence, messenger RNA stability, and DNA recognition elements of cys-14, the structural gene for sulfate permease II in Neurospora crassa.

The complete nucleotide sequence of the cys-14 gene which encodes sulfate permease II, a member of the sulfur regulatory circuit, is presented. The cys-14 gene contains four introns with consensus splice site sequences and is transcribed from four closely spaced initiation sites located approximately 20 bp upstream of the ATG initiation codon. The translated CYS14 protein is composed of 781 amino acids with a molecular weight of 87,037 and contains 12 potential hydrophobic membrane-spanning domains. cys-4 mRNA was found to turn over with a half-life of approximately 15 min, which presumably contributes to the regulation of sulfate permease II function. The cys-14 gene is highly expressed, but only in cells subject to sulfur limitation, and is turned on by the positive-acting CYS3 sulfur regulatory protein. Results are presented which show that CYS3 protein binds with higher affinity to DNA fragments which contain two or three tandem copies of a binding site sequence. Analyses of binding site specificity via mutated binding site elements showed that different regions of the partially symmetrical CYS3 binding site are important for recognition by the CYS3 regulatory protein.