Fur-DNA interactions at the bidirectional fepDGC-entS promoter region in Escherichia coli
Fur-DNA interactions at the bidirectional fepDGC-entS promoter region in Escherichia coli
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DOI：
10.1016/s0022-2836(02)00849-5
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发表时间：
2002-10-04
影响因子：
5.6
通讯作者：
McIntosh, MA
中科院分区：
文献类型：
--
作者：
Lavrrar, JL;Christoffersen, CA;McIntosh, MA
在高铁条件下，转录抑制因子Fur与一个19 bp的一致序列5‘- gataatgataatcatatc -3’结合。大肠杆菌fepDGC-entS启动子包含两个fur结合位点（FBS），偏移6bp。对该启动子区域的遗传学研究揭示了体内铁调控缺失的两个突变。一个突变改变了FBS 1的上游部分，而另一个最初是为了提高entS启动子强度而产生的突变无意中改变了FBS 2的下游部分。在这两种情况下，仍然有一个19bp的序列，根据目前的模型，应该足以进行Fur结合。这些突变对皮毛结合的影响通过体外凝胶阻滞实验和dna酶I足迹实验来检测。虽然毛皮结合的野生型DNA具有高亲和力，但其对突变体的亲和力降低，表明这两个位点都是必需的。此外，凝胶转移研究表明，毛皮启动子复合物表现出独特的结合层次结构，在毛皮浓度增加时形成不同的物种。每个凝胶移位物种结合的DNA序列使用凝胶移位/足迹耦合技术确定。本文的数据与先前发表的数据一起，提出了一种类似于转录抑制因子DtxR的Fur-DNA相互作用的新模型。该模型预测，19-bp的一致Fur算子被配置为重叠的13-mer序列，并且两个Fur二聚体从螺旋的相反面与这些序列相互作用。(C) 2002 Elsevier Science Ltd.版权所有。
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 6 bp. 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 wildtype 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. (C) 2002 Elsevier Science Ltd. All rights reserved.