A multi-axial RNA joint with a large range of motion promotes sampling of an active ribozyme conformation
A multi-axial RNA joint with a large range of motion promotes sampling of an active ribozyme conformation
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DOI：
10.1093/nar/gkz098
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发表时间：
2019-04-23
影响因子：
14.9
通讯作者：
Legault, Pascale
中科院分区：
文献类型：
--
作者：
Girard, Nicolas;Dagenais, Pierre;Legault, Pascale
研究功能性RNA中结构元件的动力学对于更好地理解其机制和设计具有新功能的RNA是重要的。在此之前，我们进行了合理的工程研究与Varkud卫星（VS）核酶，并通过修改一个关键的接吻环相互作用（KLI）的非天然发夹基板切换其特异性。我们鉴定了具有替代KLI（核糖体RNA L 88/L22和人类免疫缺陷病毒-1 TAR/TAR*）的功能性VS核酶变体，但它们显示出约低100倍的切割活性。在这里，我们表征了KLI的动力学，以将动力学特性与功能相关联，并提高设计核酶的活性。使用温度副本交换分子动力学，我们确定的VS核酶中的天然KLI支持其封闭和活性状态的构象采样，而替代KLI显示更多的限制运动。基于体外选择，具有TAR/TAR* KLI的VS核酶变体的切割活性可以通过使KLI部分失稳但增加构象取样而显著提高。我们制定了一个机制模型的底物结合，其中KLI动力学有助于形成的活性位点。我们的模型支持RNA的模块化性质，其中子域结构和动力学有助于定义与RNA功能相关的热力学和动力学。
Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed approximate to 100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.