The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg²⁺ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg²⁺ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.

Varkud 卫星核酶催化位点特异性 RNA 切割和连接，是理解 RNA 催化的重要模型系统。在这里，我们将立体特异性硫代磷酸酯取代、精确的核碱基突变和线性自由能关系测量与分子动力学、分子溶剂化理论和从头算量子力学/分子力学自由能模拟相结合，以深入了解催化作用。通过这种理论和实验的融合，我们统一了现有的结构和功能数据，以前所未有的细节揭示了催化机制，包括过渡态的质子转移程度。此外，我们提供了关键 Mg ^2+的证据在与易断裂的磷酸盐相互作用并将一般碱基鸟嘌呤锚定在亲核试剂激活位置的活性位点中。Mg ²⁺的这一新作用增加了已知催化 RNA 策略的多样性，并统一了在 Varkud 卫星、发夹和锤头核酶类中观察到的功能特征。