Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells
Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells
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DOI：
10.1016/j.cell.2019.05.029
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发表时间：
2019-07-11
期刊：
影响因子：
64.5
通讯作者：
Pertsinidis, Alexandros
中科院分区：
文献类型：
--
作者：
Li, Jieru;Dong, Ankun;Pertsinidis, Alexandros
将来自遗传学、生物化学和结构生物学的大量知识转化为细胞内生物过程的详细分子描述仍然是一个重大挑战，迫切需要更好的成像技术。例如，转录涉及RNA聚合酶II（Pol II），调节因子（RF）和染色质之间的复杂相互作用，但在其天然细胞内环境中可视化这些动态分子交易仍然难以捉摸。在这里，我们使用纳米显微镜技术放大单个标记的基因，包括一个主动的目标锁定，超灵敏的系统，使单分子检测在可寻址的子衍射体积，在拥挤的细胞内环境。我们的图像，跟踪和量化Pol II与单分子分辨率，揭示其在转录周期中的动态。进一步探索多个功能相关的事件RF-染色质相互作用，Pol II动力学，和新生转录动力学揭示了详细的操作参数的基因调控机制迄今为止，在体内看不见的。我们的方法为活细胞中复杂分子过程的单分子研究奠定了基础。
Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hither-to-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.