Rearrangements of relative positions of core strands and helices in this unlocked state lead to the native-like topology, after which refolding of the peripheral tertiary contacts locks in the N state ( 3, 7– 10). The transition from the M state to the N state then cannot occur through a fast local rearrangement because of stereochemical constraints but instead requires unfolding of the ribozyme’s peripheral regions to release constraints on movement of core elements. A potential resolution to the paradox comes from a proposal that M and N are topologically distinct, with at least one pair of strands crossed in the core ( 7). Nevertheless, it has remained paradoxical that the transition from M to N is so slow when the states are so structurally similar to each other. Biochemical data indicate that the secondary and tertiary structures of M bear a close resemblance to N. The N state comprises an intricate catalytic core involving numerous strands and helices that bind the substrates of the splicing reaction along with an extensive ring of peripheral elements that “locks in” this core ( 6). 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. 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. 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. Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. 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). The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding.
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