Formation of Tertiary Interactions during rRNA GTPase Center Folding.
Formation of Tertiary Interactions during rRNA GTPase Center Folding.
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rRNA GTPase 中心折叠过程中三级相互作用的形成。
DOI：
10.1016/j.jmb.2015.07.013
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发表时间：
2015
影响因子：
5.6
通讯作者：
Hall,KathleenB
中科院分区：
文献类型：
--
作者：
Rau,MichaelJ;Welty,Robb;TomStump,W;Hall,KathleenB
23S rRNA的60ntGTPase中心(GAC)具有系统发育保守的二级结构，有两个发夹环和一个三向连接。当添加镁离子时，它折叠成一个复杂的三级结构，这是由共晶结构中的L11蛋白稳定的。在这里，我们通过观察对共晶体中GAC三级结构有特定作用的核碱基来监测其三级折叠和镁离子依赖的中间态的动力学。荧光碱基2-氨基嘌呤取代了三个单独的腺嘌呤，其中两个产生了远程堆积作用，一个也形成了氢键。每个地点都揭示了对镁离子添加和温度的独特响应，反映了其从二级结构到三级结构的环境变化。停流荧光实验表明，添加氯化镁后的三级结构形成动力学也是位置特异的，局部构象变化从5毫秒到4毫秒发生，S从1毫秒到5毫秒，全局折叠从1毫秒到5毫秒。通过核磁共振实验观察到稳定的氢键形成的15N碱基定点取代。平衡滴定实验表明，在化学计量浓度下，存在一个稳定的折叠中间体，并表明存在两个镁离子缔合的初始位置。
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 MgCl2are 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 with15N-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.