Determining the Mg2+ stoichiometry for folding an RNA metal ion core
Determining the Mg2+ stoichiometry for folding an RNA metal ion core
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DOI：
10.1021/ja051422h
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发表时间：
2005-06-15
影响因子：
15
通讯作者：
Herschlag, D
中科院分区：
文献类型：
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作者：
Das, R;Travers, KJ;Herschlag, D
RNA 分子的折叠和催化功能取决于它们与二价金属离子（例如镁）的相互作用。与每个分子过程一样，理解 RNA 与其金属离子的密切关系所需的最基本知识是相互作用的化学计量。不幸的是，与展开和折叠 RNA 状态相关的二价离子数量的清单一直无法获得。一种常见的方法是将适合折叠平衡的希尔系数解释为折叠时结合的金属离子的数量。然而，由于动态离子气氛中存在弥散相关的二价离子以及沿着折叠路径的多次跃迁的可能性，该方法被削弱。我们证明，使用摩尔浓度的背景单价盐可以减轻这些并发症。这些简化的溶液条件允许精确测定参与折叠四膜虫组I核酶的P4-P6结构域的金属离子核心的镁离子的化学计量。羟基自由基足迹数据的 Hill 分析表明，P4−P6 RNA 核心在两个金属离子结合时协同折叠。通过荧光滴定和原子发射光谱对与 P4−P6 RNA 相关的镁离子进行计数，有力地支持了这种出乎意料的小化学计量。通过精确定位金属离子的化学计量，这些测量为金属离子结合和 RNA 折叠之间的耦合的热力学剖析提供了关键但之前缺失的步骤。
The folding and catalytic function of RNA molecules depend on their interactions with divalent metal ions, such as magnesium. As with every molecular process, the most basic knowledge required for understanding the close relationship of an RNA with its metal ions is the stoichiometry of the interaction. Unfortunately, inventories of the numbers of divalent ions associated with unfolded and folded RNA states have been unattainable. A common approach has been to interpret Hill coefficients fit to folding equilibria as the number of metal ions bound upon folding. However, this approach is vitiated by the presence of diffusely associated divalent ions in a dynamic ion atmosphere and by the likelihood of multiple transitions along a folding pathway. We demonstrate that the use of molar concentrations of background monovalent salt can alleviate these complications. These simplifying solution conditions allow a precise determination of the stoichiometry of the magnesium ions involved in folding the metal ion core of the P4−P6 domain of theTetrahymenagroup I ribozyme. Hill analysis of hydroxyl radical footprinting data suggests that the P4−P6 RNA core folds cooperatively upon the association of two metal ions. This unexpectedly small stoichiometry is strongly supported by counting magnesium ions associated with the P4−P6 RNA via fluorescence titration and atomic emission spectroscopy. By pinpointing the metal ion stoichiometry, these measurements provide a critical but previously missing step in the thermodynamic dissection of the coupling between metal ion binding and RNA folding.