Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis
Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis
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DOI：
10.1021/bi980633e
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发表时间：
1998-08-11
期刊：
影响因子：
2.9
通讯作者：
Cech, TR
中科院分区：
文献类型：
--
作者：
Szewczak, AA;Podell, ER;Cech, TR
来自四膜虫自剪接组I内含子的P4-P6结构域RNA是一个独立的三级结构单元，在动力学折叠途径中，在其余内含子之前折叠，然后稳定其余内含子的三级结构。我们采用温度梯度凝胶电泳（TGGE）来检测P4-P6三级结构的展开。在0.9 mM Mg2+中，P4-P6的整体第三系褶皱的熔化温度约为40℃，在60℃时完全展开。在此条件下，P4-P6的折叠热力学参数为δ H′= -28 +/- 3 kcal/mol, δ S′= -91 +/- 8 eu。使用二甲基硫酸盐和CMCT对P4-P6三级结构进行化学探测证实，这些TGGE实验监测了区域整体三级褶皱的展开，并且二级结构在此温度范围内基本上不受影响。因此，与熵驱动的P1对接和鸟苷结合步骤不同，四膜虫I族内含子自剪接具有正或零的δ H项，P4-P6三级结构的形成由负的δ H项稳定。这意味着有利焓的氢键形成、核苷酸碱基堆叠和/或Mg2+在折叠结构中的结合是稳定P4-P6结构域的原因。
The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic folding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure, We have employed temperature gradient gel electrophoresis (TGGE) to examine the unfolding of the tertiary structure of P4-P6. In 0.9 mM Mg2+ the global tertiary fold of the molecule has a melting temperature of approximately 40 degrees C and is completely unfolded by 60 degrees C, Calculated thermodynamic parameters for folding of P4-P6 are Delta H degrees' = -28 +/- 3 kcal/mol and Delta S degrees' = -91 +/- 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfate and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary structure is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetrahymena group I intron self-splicing, which have positive or zero Delta H terms, P4-P6 tertiary structure formation is stabilized by a negative Delta H term. This implies that enthalpically favorable hydrogen bond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain.