Chyży Piotr, Kulik Marta, Re Suyong, Sugita Yuji, Trylska Joanna
Centre of New Technologies, University of Warsaw, Warsaw, Poland.
Faculty of Physics, University of Warsaw, Warsaw, Poland.
Front Mol Biosci. 2021 Feb 18;8:633130. doi: 10.3389/fmolb.2021.633130. eCollection 2021.
Short, structured fragments of non-coding mRNA may act as molecular switches upon binding specific ligands, regulating the translation of proteins encoded downstream this mRNA sequence. One switch, called riboswitch N1, is regulated by aminoglycosides such as neomycin. Nucleobase mutations in the apical loop, although distant from the binding pocket, significantly affect neomycin affinity and riboswitch regulatory efficiency. To explain this influence, we conducted molecular dynamics simulations using generalized replica exchange with solute tempering (gREST). Translation assay of a reporter protein in a yeast system shows that mutating A17 to G in the riboswitch apical loop reduces 6-fold the translation regulation efficiency of the mutant. Indeed, simulations of the unbound riboswitch show that G17 frequently stacks with base 7, while base 8 is stabilized towards the binding site in a way that it may interfere with the conformational selection mechanism and decrease riboswitch regulatory activity. In the riboswitch complexes, this single-point A to G mutation disrupts a strong hydrogen bond between nucleotides 5 and 17 and, instead, a new hydrogen bond between residue 17 and neomycin is created. This change forces neomycin to occupy a slightly shifted position in the binding pocket, which increases neomycin flexibility. Our simulations of the U14C mutation suggest that the riboswitch complex with neomycin is more stable if cytosine 14 is protonated. A hydrogen bond between the RNA phosphate and protonated cytosine appears as the stabilizing factor. Also, based on the cell-free translation assay and isothermal titration calorimetry experiments, mutations of nucleotides 14 and 15 affect only slightly the riboswitch ability to bind the ligand and its activity. Indeed, the simulation of the unbound U15A mutant suggests conformations preformed for ligand binding, which may explain slightly higher regulatory activity of this mutant. Overall, our results corroborate the and experiments on the N1 riboswitch-neomycin system, detail the relationship between nucleobase mutations and RNA dynamics, and reveal the conformations playing the major role in the conformational selection mechanism.
非编码mRNA的短结构片段在结合特定配体后可能充当分子开关，调节该mRNA序列下游编码的蛋白质的翻译。一种称为核糖开关N1的开关受新霉素等氨基糖苷类药物调节。顶环中的核碱基突变虽然距离结合口袋较远，但会显著影响新霉素亲和力和核糖开关调节效率。为了解释这种影响，我们使用溶质回火广义副本交换（gREST）进行了分子动力学模拟。酵母系统中报告蛋白的翻译分析表明，将核糖开关顶环中的A17突变为G会使突变体的翻译调节效率降低6倍。事实上，未结合的核糖开关的模拟表明，G17经常与碱基7堆积，而碱基8以可能干扰构象选择机制并降低核糖开关调节活性的方式向结合位点稳定。在核糖开关复合物中，这种单点A到G的突变破坏了核苷酸5和17之间的强氢键，相反，在残基17和新霉素之间形成了新的氢键。这种变化迫使新霉素在结合口袋中占据稍微偏移的位置，这增加了新霉素的灵活性。我们对U14C突变的模拟表明，如果胞嘧啶14质子化，与新霉素的核糖开关复合物会更稳定。RNA磷酸基团与质子化胞嘧啶之间的氢键似乎是稳定因素。此外，基于无细胞翻译分析和等温滴定量热法实验，核苷酸14和15的突变仅轻微影响核糖开关结合配体的能力及其活性。事实上，未结合的U15A突变体的模拟表明存在为配体结合预先形成的构象，这可能解释了该突变体略高的调节活性。总体而言，我们的结果证实了关于N1核糖开关 - 新霉素系统的 和 实验，详细阐述了核碱基突变与RNA动力学之间的关系，并揭示了在构象选择机制中起主要作用的构象。
