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首页> 外文期刊>Journal of Molecular Biology >The long-range P3 helix of the tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations
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The long-range P3 helix of the tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations

机译：四膜虫核酶的远距离P3螺旋在天然构象和错折叠构象之间的折叠过程中被破坏

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摘要

RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, we constructed 16 mutations to strengthen or weaken P3. Catalytic activity and in-line probing showed that nearly all of the mutants form the M state before folding to N. The P3-weakening mutations accelerated refolding from M (3- to 30-fold) and the P3-strengthening mutations slowed refolding (6- to 1400-fold), suggesting that P3 indeed unwinds transiently. Upon depletion of Mg2 +, the mutations had analogous effects on unfolding from N to intermediates that subsequently fold to M. The magnitudes for the P3-weakening mutations were larger than in refolding from M, and small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3 is disrupted subsequently. These results are consistent with previous results indicating unfolding of native peripheral structure during refolding from M, which probably permits rearrangement of the core. Together, our results demonstrate that exchange of the native and misfolded conformations requires loss of a core helix in addition to peripheral structure. Further, the results strongly suggest that misfolding arises from a topological error within the ribozyme core, and a specific topology is proposed.
机译：RNA易于错误折叠，但是如何正确折叠和形成错误折叠的结构仍未完全了解。四膜虫的第I组内含子核酶在体外折叠成长寿的错折叠构象（M），该构象包括广泛的天然结构，但建议其拓扑结构不同于天然状态（N）。一个领先的模型预测，拓扑结构的交换需要展开远程核心螺旋P3，尽管两种构象中都存在P3。为了测试该模型，我们构建了16个突变来增强或减弱P3。催化活性和在线检测表明，几乎所有突变体在折叠成N之前都形成M状态。P3弱化突变加速了M的重折叠（3到30倍），而P3加强突变加速了重折叠（6） -到1400倍），表明P3确实暂时消失。 Mg2 +耗竭后，这些突变对从N到随后折叠为M的中间体的折叠具有相似的影响。P3弱化突变的幅度大于从M折叠的幅度，并且小角度X射线散射表明核酶迅速扩展到中间体，随后P3被破坏。这些结果与先前的结果一致，该先前的结果表明在从M重折叠过程中天然外围结构的展开，这可能允许核心的重排。在一起，我们的结果表明天然和错误折叠的构象的交换需要除了外围结构的核心螺旋的损失。此外，该结果强烈提示错误折叠是由核酶核心内的拓扑错误引起的，并提出了特定的拓扑。

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