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Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme
Nature Structural & Molecular Biology ( IF 16.8 ) Pub Date : 2017-09-11 00:00:00 , DOI: 10.1038/nsmb.3466
Gavin J Knott 1 , Alexandra East-Seletsky 1 , Joshua C Cofsky 1 , James M Holton 2, 3, 4 , Emeric Charles 1 , Mitchell R O'Connell 1 , Jennifer A Doudna 1, 2, 5, 6, 7
Affiliation  

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.

中文翻译:

靶向 RNA 的 A 切割 CRISPR–Cas13a 酶的指导结合结构

CRISPR 适应性免疫系统通过部署 CRISPR RNA (crRNA) 引导的酶来识别和切割外源核酸,从而保护细菌免受感染。VI-A 型 CRISPR–Cas 系统包括 Cas13a 酶,这是一种 RNA 激活的 RNase,能够在靶标-转录本结合后进行 crRNA 加工和单链 RNA 降解。在这里,我们展示了与 crRNA 结合的毛螺菌科细菌的 2.0 Å 分辨率晶体结构Cas13a (LbaCas13a),代表最近发现的一种 Cas13a 酶亚型。这种结构和伴随的生化实验定义了直接导致 crRNA 成熟的 Cas13a 催化残基。此外,蛋白质内部的外源靶 RNA 指定序列的方向解释了 Cas13a 核酸酶激活的构象门控。这些结果描述了 Cas13a 酶如何产生功能性 crRNA,以及在目标 RNA 识别之前催化活性如何被阻断,这对细菌免疫和诊断应用都有影响。
更新日期:2017-09-15
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