Ligand-mediated and tertiary interactions cooperatively stabilize the P1 region in the guanine-sensing riboswitch

Riboswitches are genetic regulatory elements that control gene expression depending on ligand binding. The guanine-sensing riboswitch (Gsw) binds ligands at a three-way junction formed by paired regions P1, P2, and P3. Loops L2 and L3 cap the P2 and P3 helices and form tertiary interactions. Part of P1 belongs to the switching sequence dictating the fate of the mRNA. Previous studies revealed an intricate relationship between ligand binding and presence of the tertiary interactions, and between ligand binding and influence on the P1 region. However, no information is available on the interplay among these three main regions in Gsw. Here we show that stabilization of the L2-L3 region by tertiary interactions, and the ligand binding site by ligand binding, cooperatively influences the structural stability of terminal base pairs in the P1 region in the presence of Mg2+ ions. The results are based on molecular dynamics simulations with an aggregate simulation time of ~10 μs across multiple systems of the unbound state of the Gsw aptamer and a G37A/C61U mutant, and rigidity analyses. The results could explain why the three-way junction is a central structural element also in other riboswitches and how the cooperative effect could become contextual with respect to intracellular Mg2+ concentration. The results suggest that the transmission of allosteric information to P1 can be entropy-dominated.

核糖开关（Riboswitches）是一类依赖配体结合调控基因表达的遗传调控元件。鸟苷感应核糖开关（Gsw）通过由配对区域P1、P2与P3构成的三向连接结构结合配体。L2与L3环分别封端P2和P3螺旋，并形成三级相互作用。P1的部分序列属于决定信使RNA（mRNA）命运的开关序列。既往研究表明，配体结合与三级相互作用的存在之间存在复杂关联，同时配体结合也会对P1区域产生影响。然而目前尚无针对Gsw中这三个核心区域之间相互作用的相关研究。本研究发现，在镁离子（Mg²+）存在的条件下，三级相互作用对L2-L3区域的稳定作用与配体结合对配体结合位点的稳定作用，二者协同影响P1区域末端碱基对的结构稳定性。本研究结果基于对Gsw适配体未结合状态及G37A/C61U突变体的多个体系开展的分子动力学模拟（总模拟时长约10 μs）与刚性分析。该研究结果不仅解释了为何三向连接结构同样是其他核糖开关的核心结构元件，还阐明了这种协同效应如何随细胞内镁离子浓度呈现情境特异性变化。研究结果同时表明，别构信息向P1区域的传递过程主要由熵主导。