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Nature:CRISPR系统中Cascade复合物结构解析

标签:Cascade CRISPR RNA
摘要 : 中科院生物物理研究所王艳丽研究组在新研究中,揭示了Cascade的晶体结构及其与RNA相互作用的方式。相关文章发表于2014年8月12日的《Nature》杂志上。

 中科院生物物理研究所王艳丽研究组在新研究中,揭示了Cascade的晶体结构及其与RNA相互作用的方式。相关文章发表于2014年8月12日的《Nature》杂志上。

成簇的、有规律间隔的短回文重复序列(clustered regularly interspaeed short palindromic repeats,CRISPR)和它的辅助蛋白(CRISPR-associated, Cas)构成CRISPR/Cas系统,以一种类似于真核生物RNA干扰(RNAi)的作用机制,在原核生物抵抗入侵的噬菌体和质粒的防御系统中发挥着重要作用。CRISPR-Cas系统分为三个类型(I型,II型和III型),大肠杆菌CRISPR/Cas 系统属于I-E型,由5种Cas蛋白组成的11个亚基(其中含有1个CasA,2个CasB,6个CasC,1个CasD和1个CasE)以及一段61个核苷酸的成熟crRNA(CRISPR RNA)组成Cascade(CRISPR-associated complex for antiviral defense)复合物。Cascade复合物的质量约为405kDa,外观上呈现出近似于“海马”的结构,王艳丽研究组通过深入研究,获得了分辨率为3.05 Å的X射线晶体结构,详见下图:

该结构显示,61个核苷酸的crRNA横跨Cascade的11个亚基,并与6个CasC亚基相互作用,5’和3’末端分别被CasD和CasE锚定。CrRNA的间隔区序列定位在CasC1-6亚基形成的连续的沟槽中。来自CasC2-6的5个长β发卡结构穿过crRNA。因此crRNA被分成5个片断,每个片段包括5个堆叠的碱基和一个翻转的碱基。每一个crRNA间隔区片断通过相似的方式与CasC相互作用。进一步的研究显示,crRNA不仅在靶点识别中发挥重要作用,而且也在Cascade的组装中发挥重要作用。本研究为Cascade如何发挥功能提供了重要依据。

王艳丽研究组的博士生赵宏图为本文的第一作者,该研究得到科技部、国家自然科学基金以及中国科学院战略性先导科技专项(B类)的资助。

原文摘要:

Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli

Hongtu Zhao, Gang Sheng, Jiuyu Wang, Min Wang, Gabor Bunkoczi, Weimin Gong, Zhiyi Wei & Yanli Wang

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I–E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, wher it interacts with all six CasC subunits (named CasC1–6), with its 5′ and 3′ terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1–6 subunits. The five long β-hairpins that project from individual CasC2–6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outwards-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein–protein and protein–RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.

来源: 中科院 浏览次数:75

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