Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.
Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli.
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DOI：
10.1093/nar/gkv793
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发表时间：
2015-09-30
影响因子：
14.9
通讯作者：
Wiedenheft B
中科院分区：
文献类型：
--
作者：
van Erp PB;Jackson RN;Carter J;Golden SM;Bailey S;Wiedenheft B
在细菌和古细菌中，外源DNA的短片段被整合到重复的规则间隔短回文重复序列（CRISPR）基因座中，提供了以前与外源遗传元件相遇的分子记忆。在大肠杆菌中，短CRISPR衍生的RNA被整合到称为Cascade（CRISPR相关抗病毒防御复合物）的多亚基监视复合物中。Cascade的最新结构捕获了这种海马状RNA引导的监视复合物在与DNA靶结合之前和之后的快照。在这里，我们确定了一个3.2 X射线晶体结构的级联在一个新的晶体形式，提供了深入了解双链DNA结合的机制。使用现有的结构进行的分子动力学模拟揭示了级联的尾部，主链和腹部亚基中的残基的功能作用，这些残基对于结合双链DNA是至关重要的。结构比较用于进行功能预测，并在体内和体外测试这些预测。总的来说，在这项研究中的结果揭示了潜在的机制，涉及目标诱导的构象变化，并突出残基在DNA结合和protospacer相邻的基序识别的重要性。
In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.