Baichoo Noel, Helmann John D
Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
J Bacteriol. 2002 Nov;184(21):5826-32. doi: 10.1128/JB.184.21.5826-5832.2002.
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 approximately 30 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.
铁摄取阻遏蛋白（Fur）可根据细胞内铁水平调节铁稳态基因的表达。一般来说，Fur蛋白以高亲和力与一个名为Fur框的19碱基对反向重复序列结合。对枯草芽孢杆菌Fur识别的19个操纵位点进行比对后发现，在这个19碱基对的共有序列中存在一个不同的保守15碱基对（7-1-7）反向重复序列，且出现了两次。我们通过电泳迁移率变动分析证明，这个7-1-7反向重复序列构成了Fur高亲和力结合的最小识别位点。由此得到的修订后的共有序列与Fur旁系同源蛋白PerR识别的相关7-1-7反向重复序列非常相似。我们对枯草芽孢杆菌Fur与DNA结合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Fur研究的重新解释，支持了一个模型，即19碱基对的Fur框代表了两个与DNA螺旋相反面结合的Fur二聚体的重叠识别位点。由此产生的识别复合物让人联想到功能相关蛋白DtxR所观察到的复合物。与Fur一样，DtxR也包含一个螺旋-转角-螺旋DNA结合基序，识别一个19碱基对的反向重复序列，并且具有约30碱基对的典型DNase I足迹。通过设想Fur类似的DNA识别模式，我们可以解释先前在Fur框内注意到的内部对称性、Fur以序列选择性方式延伸到DNA相邻区域的趋势，以及观察到的DNA对酶和化学探针的保护模式。
