Abstract
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.
Original language | English |
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Article number | e2313332121 |
Journal | Proceedings of the National Academy of Sciences (PNAS) |
Volume | 121 |
Issue | 3 |
Pages (from-to) | e2313332121 |
ISSN | 0027-8424 |
DOIs | |
Publication status | Published - 16 Jan 2024 |
Keywords
- RNA
- cryo-EM
- fitness landscape
- polymerase
- ribozyme
- Cryoelectron Microscopy
- RNA/genetics
- DNA-Directed RNA Polymerases/genetics
- RNA-Dependent RNA Polymerase/genetics
- RNA, Catalytic/metabolism