Supercoiling Effects on Short-Range DNA Looping in <i>E</i>. <i>coli</i>

DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6–8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70–85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.

DNA-蛋白质环（DNA-protein loops）对于基因调控至关重要。大肠杆菌（Escherichia coli）乳糖（lac）操纵子由已被研究数十年的DNA-蛋白质环所调控。本研究以此模型为基础，验证负超螺旋张力可促进大肠杆菌内短程（6~8个DNA螺旋转数）阻遏环形成这一假说。大肠杆菌DNA的天然负超螺旋状态由旋转酶（gyrase）与拓扑异构酶（topoisomerase）的协同作用调控：二者分别负责引入与去除负超螺旋。本研究针对三种全局超螺旋水平各异的大肠杆菌菌株，定量测定其DNA环形成情况：野生型菌株、旋转酶突变体（gyrB226）以及拓扑异构酶突变体（ΔtopA10）。通过检测各菌株内源性lac操纵子的阻遏效果，以及对10种DNA环大小介于70~85碱基对的报告基因构建体的阻遏效果，完成了各菌株DNA环形成情况的测定。本研究数据可被最简洁地解读为支持以下假说：负超螺旋可通过活细菌内的小型DNA-蛋白质环促进基因阻遏。