Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan.
Department of Bioengineering, California Institute of Technology, Pasadena, CA, 91125, USA.
Angew Chem Int Ed Engl. 2019 Jun 3;58(23):7626-7630. doi: 10.1002/anie.201900610. Epub 2019 Apr 30.
Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4-binding sites revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single-molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA-binding proteins.
本文报道了使用高速原子力显微镜（HS-AFM）直接观察光响应转录因子融合蛋白 GAL4-VVD 与 DNA 之间的动态相互作用。观察到一系列不同的 GAL4-VVD 运动，如结合、滑动、停顿和解离。还观察到两条双链 DNA（dsDNA）之间的链间跳跃。使用含有五个 GAL4 结合位点的长底物 DNA 链进行的详细分析表明，GAL4-VVD 随机使用滑动和跳跃在 dsDNA 上移动，以快速找到特定的结合位点，然后停顿到特定的位点以形成稳定的复合物形成。这些结果表明该蛋白存在不同的构象，以实现滑动和停顿。这种具有纳米级分辨率的单分子成像系统为 DNA 结合蛋白的搜索机制提供了深入的了解。
