Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.
Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.
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DOI：
10.1261/rna.054916.115
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发表时间：
2016-06
期刊：
影响因子：
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通讯作者：
Lucks JB
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文献类型：
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作者：
Takahashi MK;Watters KE;Gasper PM;Abbott TR;Carlson PD;Chen AA;Lucks JB
反义rna介导的转录调控因子是控制基因表达和创建合成基因网络的有力工具。来自自然机制的RNA转录抑制因子被称为衰减因子，尽管其机制的复杂性使其难以设计，但它们的用途特别广泛。在这里，我们确定了一种新的结构-功能衰减器设计原则，使新的RNA转录抑制物的正向工程。利用细胞内SHAPE-Seq表征大肠杆菌中衰减子变异的结构，我们发现促进与反义rna相互作用的衰减子发夹需要内部环才能正常工作。这些衰减器变体的分子动力学模拟表明，这些内部回路赋予了结构灵活性。我们进一步观察了利用反义RNA相互作用抑制翻译的天然RNA机制的细胞结构中的发夹灵活性，证实了早期体外研究的结果。最后，我们使用内部环作为结构要求，在硅上设计了新的转录衰减器，并证明了它们在体内的功能。本研究建立了内环作为设计合成RNA基因调控因子的重要结构元件。我们预计，将细胞RNA结构和功能的实验测量与计算建模相结合，将能够快速发现多种天然和合成RNA调节剂的结构-功能设计原则。
Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators.