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-Structure paper
Title | Cryo-EM structure and functional landscape of an RNA polymerase ribozyme. |
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Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 121, Issue 3, Page e2313332121, Year 2024 |
Publish date | Jan 16, 2024 |
Authors | 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 / |
PubMed 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 ...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. |
External links | Proc Natl Acad Sci U S A / PubMed:38207080 / PubMed Central |
Methods | EM (single particle) |
Resolution | 5.0 Å |
Structure data | EMDB-40984, PDB-8t2p: |
Source |
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Keywords | RNA / Polymerase / Ribozyme / heterodimer |