Ion-dependent mobility effects of the Fusobacterium nucleatum glycine riboswitch aptamer II via site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR)
Ion-dependent mobility effects of the Fusobacterium nucleatum glycine riboswitch aptamer II via site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR)
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DOI：
10.1016/j.bbrc.2019.06.105
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发表时间：
2019-08-27
影响因子：
3.1
通讯作者：
Fanucci, Gail E.
中科院分区：
文献类型：
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作者：
Ehrenberger, Michelle A.;Vieyra, Aleida;Fanucci, Gail E.
利用连续波电子顺磁共振(CW-EPR)谱的定点自旋标记(SDSL)技术，研究了核梭杆菌(FN)甘氨酸核糖开关84核苷酸适配子II结构域折叠过程中骨架动力学的位点特异性变化。使用夹板结扎策略结合自旋标记。结果表明，在碱基配对和环区域，自旋标记结合到主链上的微分动力学。此外，在含有100 mM K+的RNA溶液中加入5 mM的生物相关浓度的镁离子，就会折叠并压缩结构，这是由自旋标记迁移率的降低而得出的。此外，当控制离子强度时，添加到RNA中的镁离子比单独添加K+的RNA在这两个位置诱导更多的折叠/更少的灵活性。甘氨酸的加入不会改变这个单线适体II的动力学，表明全长核糖开关结构可能是甘氨酸结合和诱导构象变化所必需的。这项工作加深了我们对如何利用拼接连接SDSL来研究大型动态RNA中的差异动力学的理解，为RNA折叠和结构如何由单价阳离子和二价阳离子差异稳定提供了见解。(C)2019 Elsevier Inc.保留所有权利。
Site-directed spin-labeling (SDSL) with continuous wave electron paramagnetic resonance (cw-EPR) spectroscopy was utilized to probe site-specific changes in backbone dynamics that accompany folding of the isolated 84 nucleotide aptamer II domain of the Fusobacterium nucleatum (FN) glycine riboswitch. Spin-labels were incorporated using splinted ligation strategies. Results show differential dynamics for spin-labels incorporated into the backbone at a base-paired and loop region. Additionally, the addition of a biologically relevant concentration of 5 mM Mg2+, to an RNA solution with 100 mM K+, folds and compacts the structure, inferred by a reduction in spin-label mobility. Furthermore, when controlling for ionic strength, Mg2+ added to the RNA induces more folding/less flexibility at the two sites than RNA with K+ alone. Addition of glycine does not alter the dynamics of this singlet aptamer II, indicating that the full length riboswitch construct may be needed for glycine binding and induced conformational changes. This work adds to our growing understanding of how splinted-ligation SDSL can be utilized to interrogate differential dynamics in large dynamic RNAs, providing insights into how RNA folding and structure is differentially stabilized by monovalent versus divalent cations. (C) 2019 Elsevier Inc. All rights reserved.