Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa.
Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa.
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CYS3 调节剂（一种 bZIP DNA 结合蛋白）的产生足以诱导粗糙脉孢菌中硫基因的表达。
DOI：
10.1128/mcb.12.4.1568-1577.1992
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发表时间：
1992
影响因子：
5.3
通讯作者：
Paietta,JV
中科院分区：
文献类型：
--
作者：
Paietta,JV
Thecys-3+基因突变体粗糙脉孢菌编码bZIP（碱性区域-亮氨酸拉链）调节蛋白，其对于硫结构基因表达是必需的（例如，核转录实验证实cys-3+受硫调控，在硫控制子（scon）-负调控子突变体中cys-3+的转录是组成型的。鉴于这些结果，我已经测试了在高硫（抑制）条件下cys-3+的表达是否足以诱导硫基因表达。那将crassaβ-tubulin（tub）启动子与thecys-3+编码片段融合，用于转化acys-3缺失突变体。通过北方（RNA）和Western免疫印迹分析证实了在高硫条件下生长的同核转化体中thetub：：cys-3融合的功能。Thetubzzcys-3转化体在正常抑制高硫条件下显示芳基硫酸酯酶基因表达。使用编码温度敏感性CYS 3蛋白的Atub：：cys-3 tsfusion来确认诱导的结构基因表达是由于CYS 3蛋白功能。组成型CYS 3生产在阻遏条件下不诱导escon-2+表达。此外，acys-3启动子与acZ的融合表明，CYS 3的产生足以诱导其自身的表达，并提供了体内自我调节的证据。最后，通过体外异源二聚体研究，观察到在thecys-3位点携带点突变的菌株的明显抑制作用。这些结果支持CYS 3作为转录激活因子的解释，其调节是硫限制触发的信号响应途径中的关键控制点。
Thecys-3+geneoî Neurospora crassaencodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g.,ars-1+).Nuclear transcription assays confirmed thatcys-3+was under sulfur-regulated transcriptional control and thatcys-3+transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression ofcys-3+under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. TheN. crassaβ-tubulin (tub) promoter was fused to thecys-3+coding segment and used to transform acys-3deletion mutant. Function of thetub::cys-3fusion in homokaryotic transformants grown under high-sulfiir conditions was confirmed by Northern (RNA) and Western immunoblot analysis. Thetubzzcys-3transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. Atub::cys-3tsfusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not inducescon-2+expression under repressing conditions. In addition, acys-3promoter fusion tolacZshowed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at thecys-3locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.
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