Escolar L, Pérez-Martín J, de Lorenzo V
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Madrid, 28049, Spain.
J Mol Biol. 1998 Oct 30;283(3):537-47. doi: 10.1006/jmbi.1998.2119.
The mode of DNA binding of the Fur (ferric uptake regulator) repressor which controls transcription of iron-responsive genes in Escherichia coli, has been re-examined. Using as a reference the known sites at the promoter of the aerobactin operon of Escherichia coli, we have compared in detail the patterns of interaction between the purified Fur protein and natural or synthetic DNA targets. DNase I and hydroxyl radical footprinting, as well as missing-T assays, consistently revealed that functional Fur sites are composed of a minimum of three repeats of the hexameric motif GATAAT rather than by a palindromic 19 bp target sequence. Extended binding sites, constructed by stepwise addition of one or two direct repeats of the same sequence, were occupied co-operatively by Fur with the same pattern of interactions as those observed with the core of three repeats. This indicated that functional sites with a range of affinities can be formed by the addition of discrete GATAAT extensions to a minimal recognition sequence. The fashion in which Fur binds its target, virtually unknown in prokaryotic transcriptional regulators, accounts for the observed helical wrapping of the protein around the DNA helix.
对控制大肠杆菌中铁反应基因转录的Fur（铁摄取调节因子）阻遏物的DNA结合模式进行了重新研究。以大肠杆菌气杆菌素操纵子启动子处的已知位点为参考，我们详细比较了纯化的Fur蛋白与天然或合成DNA靶标之间的相互作用模式。DNase I和羟基自由基足迹分析以及缺失-T分析一致表明，功能性Fur位点至少由六聚体基序GATAAT的三个重复序列组成，而不是由回文19bp靶序列组成。通过逐步添加一个或两个相同序列的直接重复序列构建的扩展结合位点，被Fur协同占据，其相互作用模式与在三个重复序列核心处观察到的相同。这表明，通过向最小识别序列添加离散的GATAAT延伸，可以形成具有一系列亲和力的功能性位点。Fur与其靶标结合的方式在原核转录调节因子中几乎未知，这解释了观察到的蛋白质围绕DNA螺旋的螺旋缠绕现象。
