Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum
Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum
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DOI：
10.1074/jbc.273.50.33841
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发表时间：
1998-12-11
影响因子：
4.8
通讯作者：
Crosa, JH
中科院分区：
文献类型：
--
作者：
Chai, SH;Welch, TJ;Crosa, JH
鳗弧菌775株铁转运基因fatDCBA的表达受两种铁应答阻遏物Fur蛋白和反义RNA（RNA α）的负调控。本文报道了铁转运基因启动子的鉴定，并研究了鳗弧菌毛皮蛋白与该启动子的相互作用。通过引物延伸和S1定位分析，铁转运启动子定位在fatD上游约300个碱基对的区域中。当检测启动子-lacZ融合体时，响应于野生型菌株中的铁耗竭测量启动子的高活性，而在Fur缺陷菌株中启动子是组成型的。凝胶阻滞和DNA酶I印迹分析表明，Fur特异性结合两个连续的位点，包括启动子区和转录起始位点下游的区域。鉴定的Fur结合位点彼此之间以及与大肠杆菌Fur蛋白的共有序列之间显示出较低程度的同源性。DNA酶I足迹模式表明Fur与这两个位点的顺序相互作用，使得模板链中的保护和非模板链中的核酸酶的超敏感性。超敏感位点的周期性表明，启动子DNA在与Fur结合后发生了结构变化，这可能在基因表达的抑制中起作用。
The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNA alpha. Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V. anguillarum Fur protein and this promoter. The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain. Gel retardation and DNase I fool;print analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hyper sensitivity to the nuclease in the nontemplate strand. The periodicity of the hypersensitive sites suggested that the promoter DNA undergoes a structural change upon binding to Fur, which might play a role in the repression of gene expression.