小柯机器人

转座子编码CRISPR-Cas系统靶向DNA的结构基础
2019-12-19 16:04

美国哥伦比亚大学Israel S. Fernández、Samuel H. Sternberg等研究人员合作解析了转座子编码的CRISPR-Cas系统靶向DNA的结构基础。2019年12月18日,《自然》在线发表了这项成果。

研究人员使用冷冻电镜描述了由霍乱弧菌Tn6677转座子编码的TniQ–Cascade复合物结构,并揭示了这种功能性偶联的机理基础。冷冻电镜图可以从头建模和完善转座蛋白TniQ,该蛋白在Casscade和Cas7形成的界面(临近CRISPR RNA (crRNA)3'末端附近)以头尾相接的形式与Cascade复合物结合形成二聚体。天然Cas8–Cas5融合蛋白结合5'crRNA手柄,并通过灵活的插入域与TniQ二聚体接触。靶DNA结合结构揭示了原间隔物相邻基序识别和R环形成所必需的关键相互作用。这项工作为从结构上了解TniQ–Cascade的DNA靶向如何导致下游转座酶蛋白质的下游募集奠定了基础,并将指导蛋白质工程工作以利用该系统在基因组工程应用中进行可编程DNA插入。

据悉,细菌利用CRISPR和Cas基因编码的适应性免疫系统在病原体和移动遗传元件攻击时保持基因组完整性。I型CRISPR-Cas系统通常通过核糖核蛋白复合体Cascade和解旋酶核酸酶Cas3的联合作用靶向外源DNA降解,但是缺少Cas3的核酸酶缺陷型I系统被细菌重新用于RNA引导的转座(通过类似于Tn7-RNA的转座子)。但CRISPR和转座子相关的机器如何在DNA靶向和插入过程中协同工作仍然未知。

附:英文原文

Title: Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system

Author: Tyler S. Halpin-Healy, Sanne E. Klompe, Samuel H. Sternberg & Israel S. Fernández

Issue&Volume: 2019-12-18

Abstract: Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements13. Type I CRISPRCas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicasenuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQCascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3 end of the CRISPR RNA (crRNA). The natural Cas8Cas5 fusion protein binds the 5 crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQCascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications. Cryo-electron microscopy structures of the TniQCascade complex encoded by the Vibrio cholerae Tn6677 transposon reveal the mechanistic basis of the functional association of CRISPR- and transposon-associated machineries.

DOI: 10.1038/s41586-019-1849-0

Source:https://www.nature.com/articles/s41586-019-1849-0

 

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:43.07
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

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