In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

中文翻译：

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合理设计真核核糖开关，通过体外动力学捕获机制以高转换效率上调 IRES 介导的翻译起始

一般来说，通过共转录动力学捕获机制发挥作用的核糖开关（kt-核糖开关）在响应其配体分子的实际浓度时表现出比通过平衡机制发挥作用的eq-核糖开关更高的转换效率。然而，前者由于其更复杂的机制而设计起来更加困难。我们在这里成功地开发了一种合理的策略来构建真核kt-核糖开关，其配体依赖性地增强由内部核糖体进入位点（IRES）介导的翻译起始。这是通过利用通过筛选鉴定的高效细菌 kt-核糖开关的一些预测结构特征以及通过将适体结构域与 IRES 完全解耦来实现的。通过该策略优化的三个 kt-核糖开关（每个都响应不同的配体）比之前优化的 eq-核糖开关（调节小麦胚芽提取物中相同的 IRES 介导的翻译）表现出三到七倍的诱导率（高达~90）。由于 IRES 在各种真核表达系统中都发挥着良好的作用，因此这些类型的 kt-核糖开关有望作为基于 RNA 的主要真核基因调节剂。此外，本策略还可应用于其他类型kt-核糖开关的合理构建，包括那些在细菌表达系统中发挥作用的kt-核糖开关。