Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2024-01, Vol.121 (3), p.e2313332121

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2024

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.