结构生物学。预先组织好的 Cas9 指导 RNA 复合物用于目标 DNA 识别。
STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.
机构信息
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
出版信息
Science. 2015 Jun 26;348(6242):1477-81. doi: 10.1126/science.aab1452.
Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.
细菌适应性免疫利用 CRISPR（成簇规律间隔短回文重复）相关（Cas）蛋白与 CRISPR 转录本一起降解外源 DNA。在 II 型 CRISPR-Cas 系统中，Cas9 内切酶的激活用于在指导 RNA 结合时进行 DNA 识别，其机制尚不清楚。与单指导 RNA 结合的 Cas9 的晶体结构揭示了一种与 apo 和 DNA 结合状态都不同的构象，其中最初 DNA 检测所需的 10 个核苷酸 RNA“种子”序列预先形成 A 型构象。该向导 RNA 的这一部分对于 Cas9 形成具有 DNA 识别能力的结构至关重要，该结构准备与双链 DNA 靶序列结合。我们认为这是“种子”机制的趋同进化，类似于真核生物中 Argonaute 蛋白在 RNA 干扰期间使用的机制。
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