Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
RNA. 2024 Nov 18;30(12):1646-1659. doi: 10.1261/rna.080039.124.
Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally occurring motifs, the GAAA tetraloop • tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several nonnative TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures.
折叠 RNA 包含三级接触基序，其结构和能量在不同的 RNA 中保持保守。RNA 基序的可传递性质简化了 RNA 折叠问题，但测量许多基序的能量和构象性质仍然是一个挑战。在这里，我们使用高通量热力学方法来研究序列变化如何改变天然基序（GAAA 四链体环 • 四链体环受体（TLR）相互作用）的结合特性。我们测量了 TLR 序列的结合能和构象偏好，这些序列跨越了从规范的 11ntR 到另外两个天然 TLR（IC3R 和 Vc2R）的突变途径。虽然 IC3R 和 Vc2R 具有高度相似的能量和构象特性，但映射它们从 11ntR 转换为这些特性变化的序列变化的图谱却大不相同。能量图谱的差异源于将 11ntR 转换为 IC3R 和 Vc2R 所需的突变，而不是这些 TLR 固有能量结构的差异。构象图谱具有几个非天然 TLR 变体，它们的构象偏好与初始和最终 TLR 都不同；这些物种代表了沿着多维序列空间向具有更大适应性的序列的潜在分支点，这些序列在其他具有替代构象偏好的 RNA 环境中具有更高的适应性。我们的高通量、定量方法揭示了序列适应性景观的复杂性质，并为其分子起源提供了模型。系统和定量的分子方法为理解天然 RNA 如何在复杂景观中进化提供了关键的见解，因为它们会对选择性压力做出反应。
