Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure.
Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure.
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DOI：
10.1021/ja2020923
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发表时间：
2011-08-31
影响因子：
15
通讯作者：
Draper, David E.
中科院分区：
文献类型：
--
作者：
Leipply, Desirae;Draper, David E.
一些RNA采用的折叠策略是螯合口袋或空腔中的阳离子，其中离子中和溶剂无法接触到的磷酸盐中的电荷。尽管这种掩埋的镁离子-RNA络合物可能对某些RNA三级结构中依赖于镁离子的稳定自由能的很大一部分负责，但直接测量是不可行的，因为很难找到在什么条件下镁离子螯合自由能与RNA折叠以及与其他环境中的镁离子的不利相互作用解偶联。在一个58聚的rRNA片段中，我们使用了一个高亲和力的嗜热核糖体蛋白来捕获RNA在一个几乎与天然相同的结构中；镁离子和蛋白质稳定的结构不同于位于螯合位点的单个核苷酸的溶剂暴露。在此条件下，高亲和性络合位的滴定发生在镁离子浓度的微摩尔范围内，并部分地被离子气氛中镁离子的积累所分解。从这些实验中，我们估计了在被访问的镁离子浓度范围内的总的和特定位置的镁离子-RNA相互作用自由能。在0.1mM的镁离子和60 mM的K+离子下，特定的结合部位贡献了所有来源的~−~(2+)相互作用自由能的~−~3千卡/摩尔；在较高的镁离子浓度下，结合贡献的比例变小(−4.5vs.−33千卡/摩尔)。在接近生理离子的条件下，特定的结合部位是饱和的，但只提供镁-RNA相互作用总自由能的一小部分。
A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg2+-RNA chelates could be responsible for a significant fraction of the Mg2+-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg2+ chelation is uncoupled from RNA folding and from unfavorable interactions with Mg2+ ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg2+- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg2+ concentration, and is partially resolved from the accumulation of Mg2+ in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg2+ - RNA interaction free energies over the range of accessed Mg2+ concentrations. At 0.1 mM Mg2+ and 60 mM K+, specific site binding contributes ~ −3 kcal/mol of the total Mg2+ interaction free energy of ~−13 kcal/mol from all sources; at higher Mg2+ concentrations the site-binding contribution becomes a smaller proportion of the total (−4.5 vs. −33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but provide only a fraction of the total free energy of Mg2+ - RNA interactions.
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