Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

• 成果类型: Article
• 署名作者: Mcrae, Ewan K. S.; Wan, Christopher J. K.; Kristoffersen, Emil L.; Hansen, Kalinka; Gianni, Edoardo; Gallego, Isaac; Curran, Joseph F.; Attwater, James; Holliger, Philipp; Anderse, Ebbe S.
• 署名单位: Aarhus University; MRC Laboratory Molecular Biology
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8672
• DOI: 10.1073/pnas.2313332121
• 发表日期: 2024-01-16
• 关键词: fitness landscape; polymerization; template; network; core

摘要

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-angstrom resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70 degrees angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs. Sign up for PNAS aler