期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：37
DOI：10.1073/pnas.2209146119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Taking advantage of single-particle cryogenic electron microscopy (cryo-EM) to analyze highly heterogeneous or flexible samples, we obtained long-awaited three-dimensional (3D) structures of the misfolded
Tetrahymena ribozyme. These structures provide clear evidence for a previously proposed topological isomer model, in which the stereochemically impossible crossing of two core RNA strands prevents rapid rearrangement of the misfolded state to the native state. Topological isomers may be widespread in misfolding of complex RNA, and these cryo-EM structures set a foundation for dissecting their detailed kinetic mechanisms and functional consequences in a paradigmatic model system.
The
Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M)
in vitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the
Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme’s guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5′-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.
