Weigand Julia E, Gottstein-Schmidtke Sina R, Demolli Shemsi, Groher Florian, Duchardt-Ferner Elke, Wöhnert Jens, Suess Beatrix
Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany).
Chembiochem. 2014 Jul 21;15(11):1627-37. doi: 10.1002/cbic.201402067. Epub 2014 Jun 20.
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.
合成核糖开关可作为合成生物学中复杂的基因控制装置，通过直接的RNA-配体相互作用来调节基因表达。我们分析了一种合成新霉素核糖开关，它折叠成一个茎环结构，其内部环对配体结合和调节很重要。它由一个包含U型转弯和一个突出腺嘌呤的末端六环封闭。我们通过饱和诱变、等温滴定量热法（ITC）和核磁共振（NMR）研究了末端环中序列、结构和生物活性之间的关系。与典型U型转弯折叠相对应的突变体保留了生物活性。U型转弯中堆积相互作用的改善导致了一种调节活性略有增强的RNA元件。对于U型转弯基序的第一个位置和突出碱基，观察到了最初无法根据适体-配体复合物的结构来解释的序列-活性关系。然而，对这些突变体的核磁共振研究揭示了适体在其游离或结合状态下的结构与动力学之间以及生物活性之间的微妙关系。
