Mojica F J, Higgins C F
Nuffield Department of Clinical Biochemistry, Imperial Cancer Research Fund Laboratories, University of Oxford, John Radcliffe Hospital, UK.
Mol Microbiol. 1996 Dec;22(5):919-28. doi: 10.1046/j.1365-2958.1996.01541.x.
According to the twin-supercoiled domain model, a local domain with a high level of DNA supercoiling can be generated between two divergently transcribed promoters. We have tested this model directly using the rate of DNA photo-cross-linking by 4,5', 8-trimethylpsoralen to measure local regions of unconstrained supercoiling in vivo. We demonstrate that, in plasmids, a localized domain of highly supercoiled DNA is generated upstream of the tetA promoter. The level of supercoiling of this domain depends on the activity of the tetA promoter and on the extent of translation of the tetA gene product. The highest level of supercoiling within this localized domain was observed in a topA strain, although the generation of a localized supercoiling domain could also be detected in a topA+ background, consistent with potential physiological significance. The level of supercoiling in this localized domain correlated with activation of the supercoiling-sensitive leu-500 promoter located upstream of tetA. These data provide a direct demonstration that localized domains of increased negative supercoiling can be generated upstream of an actively transcribed promoter, and that this can result in supercoiling-mediated transcriptional coupling between two promoters.
根据双超螺旋结构域模型，在两个反向转录的启动子之间可以产生一个具有高水平DNA超螺旋的局部结构域。我们通过4,5',8-三甲基补骨脂素的DNA光交联速率直接测试了该模型，以测量体内无约束超螺旋的局部区域。我们证明，在质粒中，tetA启动子上游会产生一个高度超螺旋DNA的局部结构域。该结构域的超螺旋水平取决于tetA启动子的活性以及tetA基因产物的翻译程度。在topA菌株中观察到该局部结构域内的超螺旋水平最高，尽管在topA+背景中也能检测到局部超螺旋结构域的产生，这与潜在的生理意义相符。该局部结构域中的超螺旋水平与位于tetA上游的超螺旋敏感型leu-500启动子的激活相关。这些数据直接证明，在活跃转录的启动子上游可以产生局部增加的负超螺旋结构域，并且这可能导致两个启动子之间的超螺旋介导的转录偶联。
