cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element.

Abstract

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone Hl. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.In the filamentous fungus Neurospora crassa, a high degree of genetic and metabolic regulation governs the expression of sets of enzymes within various global areas of metabolism such as nitrogen, phosphorus, and sulfur catabolism (7, 18). The sulfur control circuit of N. crassa consists of a set of unlinked structural genes which specify enzymes involved in sulfur metabolism. Synthesis of this entire family of sulfur-related enzymes, which includes aryl sulfatase, choline sulfatase, sulfate permease, a high-affinity methionine permease, and an extracellular protease occurs only when cellular levels of sulfur become limited (10,16,19,22). The expression of these sulfur-catabolic enzymes is controlled by two distinct regulatory genes. One of these, designated scon (for sulfur controller), appears to act in a negative fashion; scon mutants are insensitive to sulfur catabolite repression and thus express the sulfur-related enzymes in a constitutive fashion (4). The other sulfurregulatory gene, known as cys-3, acts in a positive manner to activate the expression of the various sulfur-related genes (17,21).Two different sulfate permease species, specified by distinct and unlinked structural genes, are both members of the sulfur circuit (16). The structural gene for sulfate permease II, cys-14, has been cloned and shown to encode an mRNA of approximately 3 kilobases (kb) whose content is highly regulated by cys-3, by scon, and by the sulfur status of the celis (11). Thus, it appears that both of the regulatory genes as well as sulfur repression act at the level of transcription or at a closely related step such as mRNA processing or stability. The cys-3 major sulfur control gene has been postulated to encode a regulatory protein which is needed to turn on the expression of the entire set of sulfur-related activities, presumably by binding at target DNA sequences adjacent to each structural gene (11). Mutants...

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

“…Binding sites for the global-acting NIT2 have been located in the promoters of the N. crassa nit-3, alc, and lao genes, which encode nitrogen catabolic enzymes (8,13). Each site contains two or more GATA core elements, which were found to be essential for NIT2 binding, although bases flanking the GATA sequences seemed to make only minor contributions to binding-site strength (1,2).…”

Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its mRNA.

“…In N. crassa, long leaders have been found mainly in mRNAs of regulatory genes: qa-iS and qa-lF (407 and 328 nucleotides, respectively [25]), nit-2 (284 nucleotides [23]), and cpc-J (720 nucleotides [47]). All might be expected to encode relatively low-abundance proteins, although the regulatory cys-3 gene, ostensibly in the same category, has a leader of only 30 nucleotides (24). A long leader per se may have evolved under selective pressure to reduce gene expression (for instance, by diminishing the probability that scanning ribosomes reach the coding sequence) more than transcriptional mechanisms alone would allow.…”

nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.

“…DNA fragments used for gel-band-mobility-shift experiments were radioactively labeled with 32P by filling-in with the Klenow fragment of DNA polymerase I. Mobility-shift experiments were carried out as described (28) The expressed protein (1 ,Ag) and a 32P-labeled DNA fragment (10,000 cpm) were incubated at room temperature for 25 min in a buffer containing 12 mM Hepes (pH 7.9), 2 mM dithiothreitol, 3.5 mM MgCl2, 50 mM KCI, 15% (vol/vol) glycerol, and 2 ,ug of poly(dI)-poly(dC) before being loaded onto the gel.…”

“…Neurospora has been utilized as a model lower eukaryote to investigate complex regulatory circuits (1)(2)(3). The nitrogen regulatory circuit of Neurospora contains a substantial number of unlinked structural genes that encode enzymes required for utilization of various secondary nitrogen sources, when primary nitrogen sources (e.g., ammonia or glutamine) are not available (1,4,5).…”