Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch
Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch
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DOI：
10.1002/cbic.201402067
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发表时间：
2014-07
期刊：
影响因子：
3.2
通讯作者：
J. Weigand;Sina R. Gottstein‐Schmidtke;Shemsi Demolli;Florian Groher;E. Duchardt-Ferner;J. Wöhnert;B. Suess
中科院分区：
文献类型：
--
作者：
J. Weigand;Sina R. Gottstein‐Schmidtke;Shemsi Demolli;Florian Groher;E. Duchardt-Ferner;J. Wöhnert;B. Suess
在合成生物学中，合成核糖开关可以作为复杂的遗传控制装置，通过直接rna -配体相互作用调节基因表达。我们分析了一个合成的新霉素核糖开关，它折叠成一个茎环结构，内部环对配体的结合和调节很重要。它由一个末端六环封闭，末端六环包含一个U - turn和一个环外腺嘌呤。我们通过饱和诱变、ITC和NMR研究了末端环的序列、结构和生物活性之间的关系。与典型U - turn折叠相对应的突变体保留了生物活性。U - turn中堆叠相互作用的改善导致RNA元件的调节活性略有增强。对于U - turn基序和环出碱基的第一个位置，我们观察到的序列-活性关系最初不能用适体-配体复合物的结构来解释。然而，这些突变体的核磁共振研究揭示了适体在其自由或结合状态下的结构和动力学与生物活性之间的微妙关系。
Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U‐turn and a looped‐out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U‐turn fold retained biological activity. An improvement of stacking interactions in the U‐turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U‐turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.