School of Chemistry, University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom.
Org Biomol Chem. 2014 Mar 7;12(9):1495-509. doi: 10.1039/c3ob41840e.
The three dimensional solution structures of a highly conserved 16mer RNA, endowed with a classic 'GNRA' tetraloop motif, and a 17mer RNA containing a cytosine-rich heptaloop which is predicted to be a potential receptor for the former RNA, of the I-domain of Encephalomyocarditis virus IRES have been determined by NMR spectroscopy. As Mg(2+) plays an important role in the activity of the IRES, the corresponding NMR structures of the Mg(2+) bound RNA complexes have also been determined. These RNA NMR structures, 16mer (21 constraints per residue), 16mer RNA/Mg(2+) (21 constraints per residue), 17mer (17 constraints per residue) and 17mer RNA/Mg(2+) (16 constraints per residue), were calculated to a high degree of precision with low RMSDs and low clash scores represent, to the best our knowledge, the first structures of a type II picornavirus IRES. Conformational analysis of the average structure showed that the RNAs and their Mg(2+) complexes adopt characteristic A-form helical structures, stabilised by canonical and non-canonical interactions in both the stem and loop regions. The GCGA tetraloop of the 16mer folds into a standard GNRA conformation, with the structural role of A550 being in the form of a G547.A550 sheared base-pair made up of two hydrogen bonds. Further, the previously uncharacterised AACCCCA heptaloop present in the 17mer forms a compact tertiary loop motif, held together by strong π-π interactions. Analysis of the NMR structures demonstrates that the role of Mg(2+) is principally to confer enhanced stability to the RNAs whereby the tetra- and heptaloops can achieve optimum conformation for any RNA-RNA interactions which are crucial for understanding the structure-function relationship of the IRES.
脑炎心肌炎病毒 IRES 的 I 结构域中具有经典“GNRA”四联体基序的高度保守 16mer RNA 和包含富含胞嘧啶的七联体环的 17mer RNA 的三维溶液结构已通过 NMR 光谱确定。由于 Mg（2+）在 IRES 的活性中起着重要作用，因此也确定了相应的 Mg（2+）结合 RNA 复合物的 NMR 结构。这些 RNA NMR 结构，16mer（每个残基 21 个约束），16mer RNA/Mg（2+）（每个残基 21 个约束），17mer（每个残基 17 个约束）和 17mer RNA/Mg（2+）（每个残基 16 个约束），以高精度计算，具有低 RMSD 和低冲突分数，代表了我们迄今为止对 II 型小核糖核酸病毒 IRES 的第一个结构。平均结构的构象分析表明，RNA 及其 Mg（2+）复合物采用特征 A 型螺旋结构，在茎和环区域均通过规范和非规范相互作用稳定。16mer 的 GCGA 四联体折叠成标准的 GNRA 构象，A550 的结构作用形式为由两个氢键组成的 G547.A550 剪接碱基对。此外，在 17mer 中存在的以前未表征的 AACCCCA 七联体环形成一个紧凑的三级环基序，通过强π-π相互作用结合在一起。NMR 结构分析表明，Mg（2+）的作用主要是赋予 RNA 增强的稳定性，从而使四联体环和七联体环能够实现任何 RNA-RNA 相互作用的最佳构象，这对于理解 IRES 的结构-功能关系至关重要。
