Specificity and regulation of DNA binding by the yeast glucose transporter gene repressor Rgt1
Specificity and regulation of DNA binding by the yeast glucose transporter gene repressor Rgt1
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DOI：
10.1128/mcb.23.15.5208-5216.2003
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发表时间：
2003-08-01
影响因子：
5.3
通讯作者：
Johnston, M
中科院分区：
文献类型：
--
作者：
Kim, JH;Polish, J;Johnston, M
Rgt 1是一种葡萄糖响应性转录因子，其结合酿酒酵母中编码葡萄糖转运蛋白的几个HXT基因的启动子并调节其响应葡萄糖的表达。Rgt 1含有一个Zn（2）Cys（6）双核簇，负责DNA结合。大多数含有该序列基序的蛋白质以二聚体形式与序列CGG的规则间隔对结合。然而，在Rgt 1调控的基因的启动子中没有具有规则间隔的CGG对，这表明Rgt 1作为单体与CGG或另一序列结合。我们鉴定了Rgt 1共有结合位点序列5 '-CGGANNA-3'，其多个拷贝存在于所有受Rgt 1调节的HXT启动子中。Rgt 1在体内以非加性、协同方式结合HXT 3启动子中的多个位点，导致HXT 3转录的协同抑制。我们发现，葡萄糖抑制Rgt 1的DNA结合能力，从而缓解HXT基因表达的抑制。Rgt 1 DNA结合活性的这种调节是由其葡萄糖诱导的磷酸化引起的：在高水平葡萄糖下生长的细胞中存在的过度磷酸化的Rgt 1在体内或体外不结合DNA;这种形式的Rgt 1在体外去磷酸化恢复其DNA结合能力。此外，当葡萄糖加入细胞并在这些条件下结合DNA时，作为组成型阻遏物发挥功能的改变的Rgt 1保持低磷酸化。这些结果表明，葡萄糖通过诱导其磷酸化调节Rgt 1的DNA结合能力。
Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose. Rgt1 contains a Zn(2)Cys(6) binuclear cluster responsible for DNA binding. Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG. However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence. We identified the Rgt1 consensus binding site sequence 5'-CGGANNA-3', multiple copies of which are present in all HXT promoters regulated by Rgt1. Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription. We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression. This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability. Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions. These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation.