Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence
Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence
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DOI：
10.1128/jb.184.21.5826-5832.2002
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发表时间：
2002-11-01
影响因子：
3.2
通讯作者：
Helmann, JD
中科院分区：
文献类型：
--
作者：
Baichoo, N;Helmann, JD
铁摄取抑制因子(Fur)蛋白调节铁稳态基因的表达，以响应细胞内的铁水平。一般来说，毛皮蛋白与一个被称为Fur box的19个碱基的反向重复序列有很高的亲和力。枯草芽孢杆菌识别的19个操作点的比对显示，在这个19个碱基的共有序列中有两个不同的保守的15个碱基(7-1-7)反向重复序列。我们使用电泳迁移率改变分析证明，这个7-1-7反向重复序列包含一个与Fur高亲和力结合的最小识别位点。所得到的修改的共识序列与PERR识别的相关7-1-7反向重复序列非常相似，PERR是一种毛皮平行序列。我们对枯草杆菌毛皮结合DNA的亲和力和化学计量学的分析，以及对先前描述的大肠杆菌毛皮研究的重新解释，支持一个模型，即19个碱基的毛皮盒代表与DNA螺旋的相反面结合的两个毛皮二聚体的重叠识别位点。由此产生的识别复合体使人想起对功能相关蛋白DtxR的识别。和毛皮一样，DtxR包含一个螺旋-转弯-螺旋DNA结合基序，识别一个19个碱基的反向重复序列，并具有一个典型的DNase I足迹，类似于30个碱基。通过设想Fur的类似DNA识别模式，我们可以解释先前在Fur框中注意到的内部对称性，Fur以序列选择性的方式延伸到DNA相邻区域的趋势，以及观察到的DNA保护模式对酶和化学探针的保护。
Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels. In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box. An alignment of 19 operator sites, recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence. We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur. The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog. Our analysis of the affinity and stoichiometry of DNA binding by B. subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix. The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR. Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of similar to30 bp. By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes.