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Folding pathways of the tetrahymena ribozyme

机译：四膜虫核酶的折叠途径

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Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs 0.6 min - 1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~ 1 min- 1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the "choice" is enforced by energy barriers that grow larger as folding progresses.
机译：像许多结构化的RNA一样，四膜虫群I内含子核酶通过多种途径和中间体折叠。在体外标准条件下，一小部分以0.6分钟-1的小穗达到原始状态（N），而其余部分则形成了长寿的错折叠构象（M），认为构象不同。这些替代结果反映了在被捕集的中间体（Itrap）破坏后，折叠后期分支的途径。在这里，我们使用催化活性来探测从Itrap到天然和错误折叠状态的折叠过渡。我们表明预测减弱核心螺旋P3的突变不会增加从Itrap折叠的速率，但它们会增加到达天然状态的比例，而不是形成折叠错误的状态。因此，P3在折叠成天然状态时被破坏，而没有折叠到错误折叠状态，并且在限速步骤之后发生P3破坏。有趣的是，增强P3的突变体也增加了天然折叠。其他实验表明，尽管这些突变体以与野生型核酶大致相同的速率常数（〜1分钟-1）达到天然状态，但它们迅速折叠成天然状态。因此，加强P3的突变体构成了一个独特的途径，该途径包括至少一个中间体，但避免了M状态，这很可能是因为P3和正确的拓扑结构是早期形成的。我们的结果突出了RNA折叠的多种途径，并说明了快速事件之间的动力学竞争如何具有长久的影响，因为“选择”是由随着折叠进展而变得越来越大的能量屏障所强制执行的。

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