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Formation of Tertiary Interactions during rRNA GTPase Center Folding

机译：rRNA GTPase中心折叠过程中三级相互作用的形成

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摘要

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2+ ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2+-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2+ addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5 ms to 4 s and with global folding from 1 to 5 s. Site-specific substitution with N-15-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2+ and suggest that there are two initial sites of Mg2+ ion association. (C) 2015 Elsevier Ltd. All rights reserved.
机译：23S rRNA的60 nt GTPase中心（GAC）具有系统发育上保守的二级结构，带有两个发夹环和一个三向连接。加入Mg2 +离子后，它会折叠成复杂的三级结构，该结构由共晶结构中的L11蛋白稳定。在这里，我们通过观察选择的核碱基来监测其三级折叠和Mg2 +依赖的中间态的动力学，这些核碱基对共晶中的GAC三级结构贡献了特定的相互作用。荧光核碱基2-氨基嘌呤取代了三个单独的腺嘌呤，其中两个进行长距离堆积相互作用，另一个还形成氢键。每个站点都显示出对Mg2 +添加和温度的独特响应，反映了其环境从二级结构到三级结构的变化。停止流荧光实验表明，添加MgCl2后形成三级结构的动力学也是位点特异性的，局部构象变化从5 ms到4 s发生，整体折叠从1到5 s。用N-15核碱基进行位点特异性取代，可以通过NMR实验观察到稳定的氢键形成。平衡滴定实验表明，稳定的折叠中间体以Mg2 +的化学计量浓度存在，并暗示有两个Mg2 +离子缔合的初始位点。 （C）2015 Elsevier Ltd.保留所有权利。

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