The osmolyte TMAO stabilizes native RNA tertiary structures in the absence of Mg2+: evidence for a large barrier to folding from phosphate dehydration.
The osmolyte TMAO stabilizes native RNA tertiary structures in the absence of Mg2+: evidence for a large barrier to folding from phosphate dehydration.
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DOI：
10.1016/j.jmb.2010.09.043
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发表时间：
2010-11-19
影响因子：
5.6
通讯作者：
Draper DE
中科院分区：
文献类型：
--
作者：
Lambert D;Leipply D;Draper DE
离子对RNA三级结构的稳定作用是众所周知的，但中性渗透剂氧化三甲胺（TMAO）也能有效地稳定RNA三级结构。为了开始理解TMAO对RNA影响的物理基础，我们对具有已知结构的五种RNA的TMAO诱导的稳定性进行了定量。所谓的m值，即在恒定KCl活性下每摩尔渗压剂的解折叠自由能增量，对于发夹二级结构为~0，对于四种RNA三级结构（30 - 86 nts）为0.70 - 1.85 kcal/mol/m。通过小角X射线散射和羟基自由基探测对两种RNA的进一步分析表明，TMAO将未折叠系综的回转半径降低到与用Mg 2+滴定中所见相同的终点，并且TMAO和Mg 2+稳定的结构是不可区分的。值得注意的是，TMAO诱导的天然构象的Mg 2+离子螯合位点形成的部分由一个掩埋的磷酸盐，即使Mg 2+是不存在的。TMAO与KCl的相互作用很弱，排除了TMAO通过增加盐活性间接稳定RNA的可能性。然而，TMAO被强烈地排除在磷酸二甲酯（钾盐的不利相互作用自由能+211 cal/mol/m）附近，磷酸二甲酯是一种模拟RNA骨架磷酸根的离子。我们认为RNA三级结构的形成伴随着大量的磷酸盐脱水（在所研究的RNA结构中损失66 - 173个水分子），TMAO主要通过减少与这种脱水相关的能量损失来起作用。我们发现TMAO和Mg 2+的影响之间的强烈相似性表明，RNA序列比特定的离子相互作用更重要，在指定的天然结构。
The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m-values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ~0 for a hairpin secondary structure and between 0.70 and 1.85 kcal/mol/m for four RNA tertiary structures (30 – 86 nts). Further analysis of two RNAs by small angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg2+, and that the structures stabilized by TMAO and Mg2+ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg2+ ion chelation site formed in part by a buried phosphate, even though Mg2+ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy +211 cal/mol/m for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66 – 173 water molecules in the RNA structures studied), and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg2+ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.
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