Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

中文翻译：

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CRISPR RNA引导的级联复合体的DNA结合和Cas3募集的构象动力学

细菌和古细菌依靠CRISPR（集群的规则间隔的短回文重复序列）RNA引导的适应性免疫系统来特异性消除外来核酸。在大肠杆菌中，短CRISPR衍生的RNA（crRNA）与Cas（与CRISPR相关的）蛋白组装成一个405公斤的多亚基监视复合体，称为Cascade（用于抗病毒防御的CRISPR相关复合体）。Cascade结合与crRNA向导互补的外源DNA，并募集Cas3，Cas3是靶标降解所需的反式核酸酶-解旋酶。级联的结构模型已经捕获了处于不同构象状态的复合物的静态快照，但是尚未测量11个亚基监视复合物的构象动力学。在这里，我们使用氢-氘交换与质谱（HDX-MS）耦合，将级联的构象动力学映射到三维结构上。来自结构动力学的新见解用于对R环协调和Cas3募集机制进行功能预测。体内和体外。我们共同展示了如何将构象动力学映射到静态3D结构上，从而为该生物机器的功能理解增加了新的维度。