Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Cantoblanco, Spain.
FEBS J. 2014 Aug;281(16):3685-700. doi: 10.1111/febs.12890. Epub 2014 Jul 14.
Internal ribosome entry site (IRES) elements are high-order RNA structures that promote internal initiation of translation to allow protein synthesis under situations that compromise the general cap-dependent translation mechanism. Picornavirus IRES elements are highly efficient elements with a modular RNA structure organization. Here we investigated the effect of Mg(2+) concentration on the local flexibility and solvent accessibility of the foot-and-mouth disease virus (FMDV) IRES element measured on the basis of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reactivity and hydroxyl radical cleavage. We have found that Mg(2+) concentration affects the organization of discrete IRES regions, mainly the apical region of domain 3, the 10 nt loop of domain 4, and the pyrimidine tract of domain 5. In support of the effect of RNA structure on IRES activity, substitution or deletion mutants of the 10 nt loop of domain 4 impair internal initiation. In addition, divalent cations affect the binding of eIF4G, a eukaryotic initiation factor that is essential for IRES-dependent translation that interacts with domain 4. Binding of eIF4G is favored by the local RNA flexibility adopted at low Mg(2+) concentration, while eIF4B interacts with the IRES independently of the compactness of the RNA structure. Our study shows that the IRES element adopts a near-native structure in the absence of proteins, shedding light on the influence of Mg(2+) ions on the local flexibility and binding of eIF4G in a model IRES element.
内部核糖体进入位点（IRES）元件是高级别的 RNA 结构，可促进翻译的内部起始，从而在影响通用帽依赖性翻译机制的情况下允许蛋白质合成。微小核糖核酸病毒 IRES 元件是具有模块化 RNA 结构组织的高效元件。在这里，我们研究了镁（Mg）2+浓度对基于选择性 2'-羟基酰化分析的引物延伸（SHAPE）反应性和羟基自由基切割测量的口蹄疫病毒（FMDV）IRES 元件局部柔韧性和溶剂可及性的影响。我们发现，Mg2+浓度会影响离散 IRES 区域的组织，主要是结构域 3 的顶部区域、结构域 4 的 10nt 环和结构域 5 的嘧啶区。为了支持 RNA 结构对 IRES 活性的影响，结构域 4 的 10nt 环的取代或缺失突变会损害内部起始。此外，二价阳离子会影响真核起始因子 eIF4G 的结合，eIF4G 是 IRES 依赖性翻译所必需的，与结构域 4 相互作用。在低 Mg2+浓度下，局部 RNA 柔韧性有利于 eIF4G 的结合，而 eIF4B 与 IRES 的相互作用独立于 RNA 结构的紧凑性。我们的研究表明，在没有蛋白质的情况下，IRES 元件采用近天然结构，这揭示了 Mg2+离子对模型 IRES 元件中局部柔韧性和 eIF4G 结合的影响。
