Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property
Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property
复制标题
茶碱核糖开关的综合表征揭示了 Shine-Dalgarno 影响激活翻译特性的两个关键特征
DOI：
10.1007/s00253-016-7988-4
复制
发表时间：
2017-03-01
影响因子：
5
通讯作者：
Zhou, Zhemin
中科院分区：
文献类型：
--
作者：
Cui, Wenjing;Cheng, Jintao;Zhou, Zhemin
生物合成元件调控的基因表达调控在生物技术和合成生物学中具有重要意义。人工合成的核糖开关是一种关键类型的元件，可以很容易地控制异源基因在不同细菌中的表达。本研究利用依赖茶碱的人工合成核糖开关及其相应的Shine-Dalgarno(SD)序列和起始密码子之间不同间距的变异体，结合枯草芽孢杆菌的强启动子apre，对其诱导和调控特性进行了全面的研究。在新构建的表达元件集合中，与间隔较长或较短的表达元件相比，具有9个碱基间距的表达元件表现出更高的表达水平、更好的诱导折叠性能和显著更低的泄漏表达。9个碱基的核糖开关表达元件在茶碱0-8 mm范围内表现出近似线性的剂量依赖关系。通过在天然序列之前插入单个A碱基来修饰SD序列能够提高诱导后的表达水平，同时降低由于升高的泄漏水平而导致的诱导倍数。具有工程SD和最佳9-BP间距的核糖开关元件表现出改变的剂量依赖关系，其中近似线性范围移动到0-4 mm，尽管它具有类似于诱导过程的轮廓。这些结果不仅为茶碱核糖开关结合枯草杆菌天然强启动子诱导表达提供了全面的数据，也为其他合成核糖开关的模块化设计提供了SD的两个关键特征。
Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.