Sulfur-regulated control of the met-2⁺ gene of Neurospora crassa encoding cystathionine ß-lyase.

BACKGROUND: Cystathionine ß-lyase performs an essential role in the transsulfuration pathway by its primary reaction of forming homocysteine from cystathionine. Understanding how the Neurospora crassa met-2⁺ gene, which encodes cystathionine ß-lyase, is regulated is important in determining the basis of the cellular control of transsulfuration. The aim of this study was to determine the nature of a potential regulatory connection of met-2⁺ to the Neurospora sulfur regulatory network. FINDINGS: The cystathionine ß-lyase (met-2⁺) gene was cloned by the identification of a cosmid genomic clone capable of transforming a met-2 mutant to methionine prototrophy and subsequently characterized. The gene contains a single intron and encodes a protein of 457 amino acids with conserved residues predicted to be important for catalysis and pyridoxal-5'-phosphate co-factor binding. The expression of met-2⁺ in wild-type N. crassa increased 3.1-fold under sulfur-limiting growth conditions as compared to the transcript levels seen under high sulfur growth conditions (i.e., repressing conditions). In a Δcys-3 strain, met-2⁺ transcript levels were substantially reduced under either low- or high-sulfur growth conditions. In addition, the presence of CYS3 activator binding sites on the met-2⁺ promoter was demonstrated by gel mobility shift assays. CONCLUSIONS: In this report, we demonstrate the sulfur-regulated expression of the met-2⁺ gene and confirm its connection to the N. crassa sulfur regulatory circuit by the reduced expression observed in a Δcys-3 mutant and the in vitro detection of CYS3 binding sites in the met-2⁺ promoter. The data further adds to our understanding of the regulatory dynamics of transsulfuration.

Analysis of the sulfur-regulated control of the cystathionine γ-lyase gene of Neurospora crassa.

BACKGROUND: Cystathionine γ-lyase plays a key role in the transsulfuration pathway through its primary reaction of catalyzing the formation of cysteine from cystathionine. The Neurospora crassa cystathionine γ-lyase gene (cys-16(+)) is of particular interest in dissecting the regulation and dynamics of transsulfuration. The aim of this study was to determine the regulatory connection of cys-16(+) to the Neurospora sulfur regulatory network. In addition, the cys-16(+) promoter was characterized with the goal of developing a strongly expressed and regulatable gene expression tool. FINDINGS: The cystathionine γ-lyase cys-16(+) gene was cloned and characterized. The gene, which contains no introns, encodes a protein of 417 amino acids with conserved pyridoxal 5'-phosphate binding site and substrate-cofactor binding pocket. Northern blot analysis using wild type cells showed that cys-16(+) transcript levels increased under sulfur limiting (derepressing) conditions and were present only at a low level under sulfur sufficient (repressing) conditions. In contrast, cys-16(+) transcript levels in a Δcys-3 regulatory mutant were present at a low level under either derepressing or repressing conditions. Gel mobility shift analysis demonstrated the presence of four CYS3 transcriptional activator binding sites on the cys-16(+) promoter, which were close matches to the CYS3 consensus binding sequence. CONCLUSIONS: In this work, we confirm the control of cystathionine γ-lyase gene expression by the CYS3 transcriptional activator through the loss of cys-16(+) expression in a Δcys-3 mutant and through the in vitro binding of CYS3 to the cys-16(+) promoter at four sites. The highly regulated cys-16(+) promoter should be a useful tool for gene expression studies in Neurospora.

DNA-binding specificity of the CYS3 transcription factor of Neurospora crassa defined by binding-site selection.

The CYS3 transcription factor is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of a coordinately regulated group of genes involved in the acquisition and utilization of sulfur in Neurospora crassa. An approach of using binding-site selection from random-sequence oligonucleotides was used to define CYS3-binding specificity. The derived consensus-binding site of ATGGCGCCAT defines a symmetrical sequence (half-site A T G/t G/a C/t) that resembles that of other bZIP proteins such as CREB and C/EBP. By comparison, CYS3 shows a greater range of binding to a central core of varied Pur-Pyr-Pur-Pyr sequences than CREB as determined by gel shift assays. The derived CYS3 consensus binding sequence was further validated by demonstrating in vivo sulfur regulation using a heterologous promoter construct. The CYS3-binding site data will be useful for the genome-wide study of sulfur-regulated genes in N. crassa, which has served as a model fungal sulfur control system.

Cloning and characterization of scon-3+, a new member of the Neurospora crassa sulfur regulatory system.

The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators. Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator. In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Deltacys-3 mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. The N. crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N. crassa sulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.