Reveal Brad S, Paietta John V
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
BMC Res Notes. 2012 Jul 2;5:339. doi: 10.1186/1756-0500-5-339.
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.
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.
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.
胱硫醚γ-裂合酶在转硫途径中发挥关键作用，其主要反应是催化胱硫醚生成半胱氨酸。粗糙脉孢菌的胱硫醚γ-裂合酶基因（cys-16(+)）对于剖析转硫途径的调控和动态变化尤为重要。本研究旨在确定cys-16(+)与粗糙脉孢菌硫调节网络的调控联系。此外，对cys-16(+)启动子进行了表征，目的是开发一种强表达且可调控的基因表达工具。
克隆并表征了胱硫醚γ-裂合酶cys-16(+)基因。该基因不含内含子，编码一个由417个氨基酸组成的蛋白质，具有保守的磷酸吡哆醛结合位点和底物-辅因子结合口袋。使用野生型细胞进行的Northern印迹分析表明，在硫限制（去阻遏）条件下，cys-16(+)转录水平升高，而在硫充足（阻遏）条件下仅以低水平存在。相比之下，Δcys-3调节突变体中的cys-16(+)转录水平在去阻遏或阻遏条件下均处于低水平。凝胶迁移率变动分析表明，cys-16(+)启动子上存在四个CYS3转录激活因子结合位点，它们与CYS3共有结合序列高度匹配。
在本研究中，我们通过Δcys-3突变体中cys-16(+)表达的缺失以及CYS3在体外与cys-16(+)启动子四个位点的结合，证实了CYS3转录激活因子对胱硫醚γ-裂合酶基因表达的调控。高度受调控的cys-16(+)启动子应是粗糙脉孢菌基因表达研究的有用工具。
