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

TY - JOUR

T1 - Cryo-EM structure and functional landscape of an RNA polymerase ribozyme

AU - McRae, Ewan K S

AU - Wan, Christopher J K

AU - Kristoffersen, Emil L

AU - Hansen, Kalinka

AU - Gianni, Edoardo

AU - Gallego, Isaac

AU - Curran, Joseph F

AU - Attwater, James

AU - Holliger, Philipp

AU - Andersen, Ebbe S

PY - 2024/1/16

Y1 - 2024/1/16

N2 - 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.

AB - 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.

KW - RNA

KW - cryo-EM

KW - fitness landscape

KW - polymerase

KW - ribozyme

KW - Cryoelectron Microscopy

KW - RNA/genetics

KW - DNA-Directed RNA Polymerases/genetics

KW - RNA-Dependent RNA Polymerase/genetics

KW - RNA, Catalytic/metabolism

U2 - 10.1073/pnas.2313332121

DO - 10.1073/pnas.2313332121

M3 - Journal article

C2 - 38207080

SN - 0027-8424

VL - 121

SP - e2313332121

JO - Proceedings of the National Academy of Sciences (PNAS)

JF - Proceedings of the National Academy of Sciences (PNAS)

IS - 3

M1 - e2313332121

ER -