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

Abstract

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 Δ cys-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 Δ cys-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