Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.
Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Universität Innsbruck, 6020 Innsbruck, Austria.
Nucleic Acids Res. 2019 Jul 26;47(13):7105-7117. doi: 10.1093/nar/gkz498.
The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.
HIV-1 Rev 反应元件 (RRE) RNA 元件通过与病毒蛋白 Rev 形成寡聚复合物来介导包含内含子的病毒 RNA 的核输出。茎 IIB 和附近的茎 II 三链结通过两个 Rev 分子的协同结合引发寡聚化。该 RRE 区域的构象灵活性对于 Rev 结合很重要。然而，其灵活性的性质仍然难以捉摸。在这里，我们使用 NMR 弛豫分散，包括一种用于直接观察大型 RNA 中瞬态构象状态的新策略，发现单独的茎 IIB 或作为较大的 RREII 三链结的一部分，与非天然的激发态（ES）构象以动态平衡的方式存在，其总种群约为 20%。ES 通过改变局部二级结构破坏 Rev 结合位点，并且通过点取代突变稳定它们会使 Rev 富含精氨酸的基序 (ARM) 的结合亲和力降低 15 到 80 倍。该集合阐明了在茎 IIB 中观察到的构象灵活性，揭示了茎 IIB 和三链结之间的远程构象耦合，这可能在协同 Rev 结合中发挥作用，并且还将非天然的 RRE 构象状态鉴定为开发抗 HIV 治疗的新靶标。
