The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5′-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5′-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.

中文翻译：

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RNA 连接酶核酶底物特异性从磷咪唑到三磷酸激活的演变


通过进化过程获得新的RNA功能对于基于RNA的原始生物学的多样化及其随后向现代生物学的转变至关重要。然而，RNA 获得新功能的机制仍不清楚。 RNA 酶是否需要全新的折叠来支持新的但相关的功能，或者重新优化活性位点就足够了？中性突变和适应性突变在进化创新中的作用是什么？在这里，我们通过关注 RNA 催化 RNA 组装中底物特异性的演变来通过实验解决这些问题。我们使用定向体外进化来证明，使用益生元相关的 5'-磷咪唑激活底物的连接酶核酶可以进化为催化与生物学相关三磷酸基团 5'-激活的底物的连接。有趣的是，尽管催化了相关反应，但新的核酶折叠成全新的结构，并通过催化 RNA 与三磷酸和磷咪唑激活底物的连接而表现出混杂性。尽管在序列和结构上不同，亲本磷咪唑化物连接酶和进化的三磷酸连接酶核酶可以通过一系列点突变连接，其中中间序列保留至少一些连接酶活性。这些不同的连接酶核酶之间存在准中性途径，表明中性漂移足以获得新的底物特异性，从而为后续的适应性优化提供机会。 从使用磷咪唑激活底物的 RNA 催化 RNA 组装到三磷酸激活底物的转变可能预示着使用单体三磷酸（三磷酸核苷，NTP）进行 RNA 合成的蛋白酶的后来进化。