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首页> 外文期刊>Journal of Molecular Biology >Fur-DNA Interactions at the Bidirectional fepDGC-entS Promoter Region in Escherichia coli.
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Fur-DNA Interactions at the Bidirectional fepDGC-entS Promoter Region in Escherichia coli.

机译：大肠杆菌双向fepDGC-entS启动子区域的Fur-DNA相互作用。

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摘要

The transcriptional repressor Fur binds to a 19-bp consensus sequence, 5'-GATAATGATAATCATTATC-3', under high iron conditions. The fepDGC-entS promoter of Escherichia coli contains two Fur-binding sites (FBS) offset by 6bp. Genetic studies of this promoter region revealed two mutations that exhibited a loss of iron regulation in vivo. One mutation altered the upstream portion of FBS 1, whereas the other, originally created to improve entS promoter strength, inadvertently altered the downstream portion of FBS 2. In both cases, there remains a 19-bp sequence that by current models should be sufficient for Fur binding. The effect of these mutations on Fur binding was examined using in vitro gel retardation assays and DNase I footprinting experiments. Though Fur bound wild-type DNA with high affinity, its affinity for the mutants was reduced, suggesting that both sites are required. In addition, gel shift studies demonstrated that the Fur-promoter complexes exhibit a unique hierarchy of binding, with distinct species forming at increasing concentrations of Fur. The DNA sequences bound in each gel-shifted species were determined using a coupled gel shift/footprint technique. The data presented here, with previously published data, suggest a new model for Fur-DNA interactions similar to that seen with the transcriptional repressor, DtxR. The model predicts that the 19-bp consensus Fur operator is configured as overlapping 13-mer sequences, and that two Fur dimers interact with these sequences from opposite faces of the helix.
机译：在高铁条件下，转录抑制因子Fur与19 bp的共有序列5'-GATAATGATAATCATTATC-3'结合。大肠杆菌的fepDGC-entS启动子包含两个Fur结合位点（FBS），相距6bp。对该启动子区域的遗传研究表明，两个突变在体内表现出铁调节的丧失。一种突变改变了FBS 1的上游部分，而另一种突变最初是为了提高entS启动子强度而创建的，无意中改变了FBS 2的下游部分。在这两种情况下，仍然存在一个19 bp的序列，根据当前模型，该序列应足以皮草装订。使用体外凝胶阻滞测定法和DNase I足迹实验检测了这些突变对Fur结合的影响。尽管Fur以高亲和力结合野生型DNA，但其对突变体的亲和力却降低了，表明这两个位点都是必需的。此外，凝胶位移研究表明，Fur-启动子复合物表现出独特的结合层次，随着Fur浓度的增加，形成了独特的物种。使用偶联的凝胶移位/印迹技术确定结合在每种凝胶移位的物种中的DNA序列。此处提供的数据与先前发表的数据一起，提出了与转录抑制子DtxR相似的Fur-DNA相互作用的新模型。该模型预测，将19 bp的共有Fur操纵子配置为重叠的13-mer序列，并且两个Fur二聚体与来自螺旋相对面的这些序列相互作用。

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