核糖开关是结合特定小分子以调节基因表达的信使RNA(mRNA)片段。合成的N1核糖开关,插入酵母mRNA控制报告基因的翻译以响应新霉素。然而,它的调节活性对单点RNA突变敏感,甚至那些远离新霉素结合位点的人。虽然新霉素与N1及其变体的关联路径仍然未知,最近的荧光动力学实验表明由构象选择驱动的两步过程。这提出了哪个步骤受突变影响的问题。为了解决这个问题,我们对N1和U14C进行了全原子二维复制交换分子动力学模拟,U14C[公式:见正文],U15A,和A17G突变体,确保RNA和新霉素的广泛构象采样。获得的新霉素缔合和结合路径,以及多维自由能概况,揭示了两步结合机制,由构象选择和诱导拟合组成。在核开关发夹中鉴定出稳定的上茎和U型转角基序后,新霉素与预先形成的N1构象结合。然而,新霉素在结合位点的定位发生在每个突变体不同的RNA-新霉素距离,这可以解释他们不同的监管活动。随后的诱导拟合源于新霉素的N3氨基与RNA的相互作用,导致G9骨干重新排列。在A17G突变体中,与N1相比,关键的C6-A17/G17堆叠在更近的RNA-新霉素距离形成。这些发现以及估计的结合自由能与实验一致,并阐明了为什么A17G突变减少而U15A增强对新霉素的N1活性。
Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C[Formula: see text], U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin\'s N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin.