Department of Biochemistry, Stanford University, Stanford, CA 94305.
Department of Chemical Engineering, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2017 Sep 12;114(37):E7688-E7696. doi: 10.1073/pnas.1703507114. Epub 2017 Aug 24.
Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔG, the probability of aligning tertiary contact partners, and ΔG, the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔG) or from changes in the electrostatic environment (ΔG) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔG). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔG values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.
几十年来，对结构 RNA 的结构和功能的研究使人们形成了这样一种观点，即 RNA 的三级结构是模块化的，由局部稳定的结构域组成，这些结构域在不同的 RNA 中保持其结构。我们提出了一个受这种模块化启发的假设，即 RNA 折叠热力学和动力学可以从复杂 RNA 各个组成部分的可分离能量贡献中进行定量预测。这种重建假设从 ΔG（排列三级接触伙伴的概率）和 ΔG（在排列状态下形成三级接触的有利能量贡献）两个方面考虑 RNA 三级折叠。该假设预测，来自不同连接螺旋和连接点的三级接触的排列变化（ΔG）或来自静电环境变化的三级接触的排列变化（ΔG）不会影响三级接触突变的能量扰动（ΔΔG）。这些预测一致表明，对模型 RNA 折叠的单分子 FRET 测量揭示了嵌入不同结构环境和不同静电条件下的三级接触突变的恒定 ΔΔG 值。这些突变的动力学效应为 RNA 元件的模块化行为提供了进一步的支持，并表明三级突变可用于识别限速步骤并剖析复杂 RNA 的折叠和组装途径。总的来说，我们的模型和结果为预测性理解 RNA 折叠奠定了基础，这将允许对 RNA 折叠热力学和动力学进行操纵。相反，本文中的方法可以识别出不能应用独立加和模型的情况，因此需要进一步研究。
