ABSTRACT

Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5ʹ-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in E. coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3ʹ-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors – a reduced fraction of mRNA in the ‘ON’ state and a considerably lower translation initiation rate in the riboswitch – contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.

摘要

核糖开关是基于RNA的调节元件，其利用配体诱导的mRNA的5′-非翻译区的结构变化来调节相关基因的表达。大多数合成核糖开关已经在基于细胞的系统中进行了选择和测试。无细胞蛋白质表达系统（CFPS）在开发和测试合成核糖开关方面具有多个优势，包括消除与复杂细胞网络的相互作用以及转录和翻译过程的解偶联。为了更好地了解核糖开关调节机制，以便更有效地优化核糖开关并将其用于生物传感应用，我们研究了茶碱响应性合成核糖开关与超级文件夹绿色荧光蛋白（sfGFP）报告基因结合的性能。大肠杆菌细胞提取物和无PURE细胞系统。为了监测mRNA的动力学，将孔雀石绿适体序列加到sfGFP mRNA的3′-非翻译区。将茶碱核糖开关的性能与组成性表达的sfGFP（对照）进行比较。在两个大肠杆菌中测试的所有茶碱浓度下，riboswitch mRNA的转录动力学与对照mRNA的转录非常相似。提取并纯CFPS。但是，即使在茶碱浓度最高的情况下，核糖开关构建体中的sfGFP表达也降低了一个数量级。建立了由动力学诱集机制控制的核糖开关激活的数学模型。与对照构建体相比，两个因素-“ ON”状态的mRNA减少和核糖开关中的翻译起始速率大大降低-导致茶碱核糖开关中蛋白质表达水平低得多。