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

Ewan K S McRae, Christopher J K Wan, Emil L Kristoffersen, Kalinka Hansen, Edoardo Gianni, Isaac Gallego, Joseph F Curran, James Attwater, Philipp Holliger, Ebbe S Andersen

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review


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-Å 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° 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.

TidsskriftProceedings of the National Academy of Sciences
Sider (fra-til)e2313332121
StatusUdgivet - 16 jan. 2024


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