Dupont J A, Snoussi K
Free University of Brussels, 1050 Brussels, Belgium.
J Biol Phys. 2009 Aug;35(3):231-43. doi: 10.1007/s10867-009-9151-2. Epub 2009 Apr 29.
NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton have been used to probe enhanced thermostability and conformational rearrangements induced by Mg(2+) in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus (EMCV) picornavirus internal ribosome entry site (IRES). We have measured some of their r(GC) base-pair lifetimes and dissociation constants under different MgCl(2) conditions, and we compare them with those of other short RNA duplexes. The RNA fragment r(CACCUGGCGACAGGUG) adopts two topologies, a palindromic duplex with two conformations and a hairpin, whose equilibrium can be monitored: the duplex form is destabilized by Mg(2+) and temperature, a delicate balance wherein the entropic contribution of the free energy helps populate the hairpin state. For both fragments, the opening rates of the r(GC) pairs are lower in the presence of Mg(2+) and their dissociation constants are smaller or comparable. Analysis of the results suggests that Mg(2+) has a preferential and specific effect on the r(CACCUGGCGACAGGUG) hairpin in the region close to the r(G*C) closing pair of the GCGA tetraloop, and the ion moves diffusively around r(GGCCAAAAGCC), thereby differentiating the GNRA and RAAA hairpin motifs that are both involved in the biological regulation functions of the EMCV IRES.
核磁共振（NMR）中来自水的磁化转移以及亚氨基质子的氨催化交换，已被用于探究镁离子（Mg²⁺）在脑心肌炎病毒（EMCV）微小核糖核酸病毒内部核糖体进入位点（IRES）的两个关键活性片段r（CACCUGGCGACAGGUG）和r（GGCCAAAAGCC）中诱导的热稳定性增强和构象重排。我们测量了它们在不同MgCl₂条件下的一些r（GC）碱基对寿命和解离常数，并将其与其他短RNA双链体的进行比较。RNA片段r（CACCUGGCGACAGGUG）呈现两种拓扑结构，一种具有两种构象的回文双链体和一种发夹结构，其平衡状态可被监测：双链体形式会因Mg²⁺和温度而不稳定，这是一种微妙的平衡，其中自由能的熵贡献有助于形成发夹状态。对于这两个片段，在Mg²⁺存在的情况下，r（GC）对的打开速率较低，且它们的解离常数较小或相当。结果分析表明，Mg²⁺对靠近GCGA四环的r（G*C）封闭对区域的r（CACCUGGCGACAGGUG）发夹具有优先且特定的作用，并且该离子在r（GGCCAAAAGCC）周围进行扩散移动，从而区分了均参与EMCV IRES生物学调节功能的GNRA和RAAA发夹基序。
