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.

Bruno protein contains an expanded RNA recognition motif.

The RNA recognition motif (or RRM) is a ubiquitous RNA-binding module present in approximately 2% of the proteins encoded in the human genome. This work characterizes an expanded RRM, which is present in the Drosophila Bruno protein, and targets regulatory elements in the oskar mRNA through which Bruno controls translation. In this Bruno RRM, the deletion of 40 amino acids prior to the N-terminus of the canonical RRM resulted in a significantly decreased affinity of the protein for its RNA target. NMR spectroscopy showed that the expanded Bruno RRM contains the familiar RRM fold of four antiparallel beta-strands and two alpha-helices, preceded by a 10-residue loop that contacts helix alpha(1) and strand beta(2); additional amino acids at the N-terminus of the domain are relatively flexible in solution. NMR results also showed that a truncated form of the Bruno RRM, lacking the flexible N-terminal amino acids, forms a stable and complete canonical RRM, so that the loss of RNA binding activity cannot be attributed to disruption of the RRM fold. This expanded Bruno RRM provides a new example of the features that are important for RNA recognition by an RRM-containing protein.