Department of Chemistry, Durham University, Durham, UK; Department of Biosciences, Durham University, Durham, UK.
Department of Biosciences, Durham University, Durham, UK.
Methods. 2023 Nov;219:68-72. doi: 10.1016/j.ymeth.2023.09.007. Epub 2023 Sep 26.
The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a "mechanical code" can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.
遗传信息的转录、复制、包装和修复普遍涉及 DNA：蛋白质相互作用和其他需要局部机械扭曲 DNA 的生物过程。因此，这种 DNA 变形过程的能量取决于 DNA 的局部机械特性，如弯曲性或扭转刚度。反过来，这些特性又取决于序列，使得序列可以通过控制 DNA 的局部机械特性来调节多种生物过程。历史上，由于缺乏技术来高通量测量基因组大片段上的局部 DNA 力学如何随序列变化，因此一直难以深入了解这种“机械密码”如何编码广泛的调控信息。最近开发的一种名为 loop-seq 的技术克服了这一难题。在这里，我们描述了 loop-seq 协议的一种变体，该变体允许在低通量下进行快速灵活性测量，而无需进行下一代测序。我们使用我们的方法验证了之前关于细菌转录因子整合宿主因子（IHF）结合位点如何作为刚性障碍的预测，该预测可以防止 IHF 结合位点所在的大肠杆菌中增强子-启动子的有效接触，而 IHF 结合可以缓解这种接触。
