Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.
Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.
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DOI：
10.1016/j.jbc.2023.104700
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发表时间：
2023-05
影响因子：
4.8
通讯作者：
Gagnon, Keith T.
中科院分区：
文献类型：
--
作者：
Sudhakar, Sruthi;Barkau, Christopher L.;Chilamkurthy, Ramadevi;Barber, Halle M.;Pater, Adrian A.;Moran, Sean D.;Damha, Masad J.;Pradeepkumar, P. I.;Gagnon, Keith T.
核糖核蛋白（RNP）包含一个或多个RNA和蛋白质分子，它们相互作用以形成稳定的复合物，通常涉及更灵活的RNA成分的构象变化，我们建议CAS12A RNP组装与其认知CRISPR RNA（CRRRNA）指南在结合更稳定的crrna 5'pseudoknot手柄的过程中，主要通过CAS12A构象变化进行序列和结构比对表明，cas12a蛋白在序列和结构上是不同的，而crrna 5'重复区域（将其折叠成伪not和锚定与Cas12a结合）是高度保守的。揭示了未结合的Apo-Cas12a的实质性灵活性。折叠。因此，指导RNA结构，以维持CRISPR防御机制的所有阶段的功能。
Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5′ pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5′ repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5′ pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5′ pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.
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