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The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations.

ABSTRACT: RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, we constructed 16 mutations to strengthen or weaken P3. Catalytic activity and in-line probing showed that nearly all of the mutants form the M state before folding to N. The P3-weakening mutations accelerated refolding from M (3- to 30-fold) and the P3-strengthening mutations slowed refolding (6- to 1400-fold), suggesting that P3 indeed unwinds transiently. Upon depletion of Mg(2+), the mutations had analogous effects on unfolding from N to intermediates that subsequently fold to M. The magnitudes for the P3-weakening mutations were larger than in refolding from M, and small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3 is disrupted subsequently. These results are consistent with previous results indicating unfolding of native peripheral structure during refolding from M, which probably permits rearrangement of the core. Together, our results demonstrate that exchange of the native and misfolded conformations requires loss of a core helix in addition to peripheral structure. Further, the results strongly suggest that misfolding arises from a topological error within the ribozyme core, and a specific topology is proposed.

SUBMITTER: Mitchell D

PROVIDER: S-EPMC3706569 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations.

Mitchell David D Jarmoskaite Inga I Seval Nikhil N Seifert Soenke S Russell Rick R

Journal of molecular biology 20130520 15

RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, ...[more]

PMID: 23702292

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Project description:Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I(trap) and misfolded (M). Here, we probe the structure and folding transition of I(trap) and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I(trap) and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3-P8 domain are more exposed to solvent in I(trap) than in M. I(trap) rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I(trap) much less than M. Thus, the rate-limiting transition from I(trap) requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I(trap). Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I(trap), whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3-P8 domain, has no effect. Thus, the unfolding required from I(trap) appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I(trap) and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.

Dataset Information

The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations.

Publications

The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations.

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