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

BMC Research Notes

John V Paietta¹  Brad S Reveal¹ 

[1] Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA

关键词: Neurospora crassa;    cys-16⁺;    CYS3 regulator;    Sulfur gene regulation;    Transsulfuration;    Cystathionine γ-lyase;   

Others  :  1166188
DOI  :  10.1186/1756-0500-5-339

 received in 2012-01-16, accepted in 2012-06-05,  发布年份 2012

【 摘 要 】

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

【 授权许可】

   
2012 Reveal and Paietta; licensee BioMed Central Ltd.

【 图 表 】

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