Shiman R, Draper D E
Department of Chemistry, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
J Mol Biol. 2000 Sep 8;302(1):79-91. doi: 10.1006/jmbi.2000.4031.
The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.
通过紫外熔解研究了单价阳离子（Li⁺、Na⁺、K⁺、Rb⁺、Cs⁺和NH₄⁺）对RNA三级结构热稳定性的影响。我们发现，对于这里使用的RNA（大肠杆菌23S rRNA的1051 - 1108位核苷酸，有四处碱基替换），单价阳离子和Mg²⁺在稳定RNA三级结构方面存在竞争，且这种竞争发生在两个界限之间：一个是Mg²⁺浓度为零的情况，另一个是其稳定作用达到最大（“饱和”）的情况。所有单价阳离子的竞争模式相同，且取决于阳离子从RNA上取代Mg²⁺的能力、在无Mg²⁺时稳定三级结构的能力以及在Mg²⁺浓度饱和时稳定三级结构的能力。单价阳离子的稳定能力取决于其未水化的离子半径，在低单价阳离子浓度和Mg²⁺饱和的情况下，当三级结构变性时，（计算得出）每个RNA分子会净释放一个单价阳离子。这意味着在这些条件下，RNA上至少有一个单价阳离子结合位点，该位点与稳定三级结构的形成特异性相关，K⁺是所测试阳离子中最有效的，而Mg²⁺在该位点似乎无效。在高离子强度且无Mg²⁺的情况下，三级结构的稳定仍然具有单价阳离子特异性且依赖于离子半径，但RNA三级结构变性时会释放更多数量的阳离子（约八个），并且在这些条件下NH₄⁺似乎是稳定三级结构最有效的阳离子。在大多数实验中，向缓冲液中加入了甲醇作为共溶剂。它的使用使得能够在单价离子作用原本过于微弱而无法观察的条件下研究其行为。甲醇可稳定RNA的三级结构而非二级结构。没有证据表明它会导致RNA阳离子结合特性发生定性变化或单价阳离子/Mg²⁺竞争模式发生改变。
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