Qin Peter Z, Feigon Juli, Hubbell Wayne L
Jules Stein Eye Institute, University of California, Los Angeles, CA 90095, USA.
J Mol Biol. 2005 Aug 5;351(1):1-8. doi: 10.1016/j.jmb.2005.06.007.
The Mg(2+)-dependent GAAA tetraloop interaction with its 11 nucleotide receptor is one of the most frequently occurring long-range tertiary interactions in RNAs. To explore conformational changes in the receptor during tetraloop docking, nitroxide spin labels were attached at each of four uridine bases, one at a time, within an RNA molecule containing the receptor sequence. In the presence of Mg2+ and the tetraloop, the electron paramagnetic resonance (EPR) spectrum of one of the labeled bases reflected a large increase in mobility, indicating unstacking of the base upon tetraloop docking. This provides direct evidence that base unstacking is an intrinsic feature of the solution tetraloop-receptor complex formed in the presence of Mg2+. Additional evidence suggests that in solution the bound receptor conformation is similar to that observed in the crystal structure of a group I intron ribozyme domain. In Mg2+ alone, a receptor conformation with an unstacked base was not detectable, suggesting that this conformation is of higher standard state free energy than that of the free receptor. This leads to the conclusion that the extensive RNA-RNA interactions observed in the crystal structure of the tetraloop-receptor complex provide larger interaction energy than the measured apparent affinity between the tetraloop and the free receptor. This is compatible with a high specificity of the tetraloop-receptor interaction.
Mg(2+)依赖的GAAA四环与其11个核苷酸受体之间的相互作用是RNA中最常见的远程三级相互作用之一。为了探索四环对接过程中受体的构象变化，在含有受体序列的RNA分子中，一次在四个尿嘧啶碱基中的每一个上连接一个氮氧化物自旋标记。在Mg2+和四环存在的情况下，其中一个标记碱基的电子顺磁共振（EPR）光谱反映出迁移率大幅增加，表明四环对接时碱基发生了堆积解聚。这提供了直接证据，表明碱基堆积解聚是在Mg2+存在下形成的溶液四环-受体复合物的一个固有特征。其他证据表明，在溶液中，结合的受体构象与在I组内含子核酶结构域的晶体结构中观察到的构象相似。仅在Mg2+存在的情况下，无法检测到具有堆积解聚碱基的受体构象，这表明这种构象的标准态自由能高于游离受体的标准态自由能。由此得出结论，在四环-受体复合物的晶体结构中观察到的广泛RNA-RNA相互作用提供的相互作用能比四环与游离受体之间测得的表观亲和力更大。这与四环-受体相互作用的高特异性是一致的。
