小柯机器人

美国哥伦比亚大学Samuel H. Sternberg、Israel S. Fernández等研究人员合作发现，选择性的TnsC招募增强RNA引导的转座保真度。相关论文于2022年8月24日在线发表在《自然》杂志上。

研究人员使用染色质免疫沉淀测序来监测了I-F型RNA引导的转座体的体内形成，从而使得研究人员能够在整合前解析不同的蛋白质招募事件。TniQ-级联复合物的DNA靶向是杂乱无章的，有数百个基因组脱靶位点被发现，但这些位点中只有一个亚群能够用于TnsC和TnsB招募，这揭示了一个关键的校对检查点。为了推进对负责转座体组装的相互作用的机理理解，研究人员用冷冻电镜确定了TnsC的结构，发现ATP结合驱动了七聚体环的形成，使DNA穿过中央孔，从而为下游整合定位了底物。总的来说，这些结果强调了在RNA引导的转位过程中，连续因子与基因组靶点结合所带来的分子特异性，并提供了一个结构路线图来指导未来的工程化。

据介绍，细菌转座子是普遍存在的移动遗传元件，使用不同的DNA结合蛋白进行水平传播。例如，大肠杆菌Tn7使用TnsD固定在一个特定的附着点上，而CRISPR相关的转座子使用I型或V型Cas效应器插入由导向RNA指定的靶点下游。尽管有这种靶向的多样性，转座总是需要TnsB，一种DDE家族的转座酶，催化DNA的切除和插入，以及TnsC，一种AAA+ATP酶，被认为是转座酶和靶向蛋白之间的交流。TnsC是如何介导这种交流，从而调节转座保真度的，至今仍不清楚。

附：英文原文

Title: Selective TnsC recruitment enhances the fidelity of RNA-guided transposition

Author: Hoffmann, Florian T., Kim, Minjoo, Beh, Leslie Y., Wang, Jing, Vo, Phuc Leo H., Gelsinger, Diego R., George, Jerrin Thomas, Acree, Christopher, Mohabir, Jason T., Fernndez, Israel S., Sternberg, Samuel H.

Issue&Volume: 2022-08-24

Abstract: Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ–Cascade complex is surprisingly promiscuous—hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

DOI: 10.1038/s41586-022-05059-4

Source: https://www.nature.com/articles/s41586-022-05059-4

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