Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation

• 成果类型: Article
• 署名作者: Dasgupta, Saurja; Weiss, Zoe; Nisler, Collin; Szostak, Jack W.
• 署名单位: Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Howard Hughes Medical Institute; Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Harvard University; Harvard Medical School; Harvard University; University of Chicago; Howard Hughes Medical Institute; University of Chicago; University of Notre Dame; University of Notre Dame; Harvard University; Harvard Medical School
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8822
• DOI: 10.1073/pnas.2407325121
• 发表日期: 2024-09-17
• 关键词: molecular recognition; nucleotide synthesis; polymerase ribozyme; amino-acids; Robustness; ligation; aptamers; complex; selection; sequence

摘要

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.