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Enhanced specificity against misfolding in a thermostable mutant of the Tetrahymena ribozyme.

ABSTRACT: Structured RNAs encode native conformations that are more stable than the vast ensembles of alternative conformations, but how this specificity is evolved is incompletely understood. Here we show that a variant of the Tetrahymena group I intron ribozyme that was generated previously by in vitro selection for enhanced thermostability also displays modestly enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. The enhanced specificity for native folding arises from mutations in two nucleotides that are close together in space in the native structure, and additional experiments show that these two mutations do not affect the stability of the misfolded conformation relative to the largely unstructured transition state ensemble for interconversion between the native and misfolded conformers. Thus, they selectively stabilize the native state, presumably by strengthening a local tertiary contact network that cannot form in the misfolded conformation. The stabilization is larger in the presence of the peripheral element P5abc, suggesting that cooperative tertiary structure formation plays a key role in the enhanced stability. The increased specificity in the absence of explicit selection suggests that the large energy gap in the wild-type RNA may have arisen analogously, a consequence of selective pressure for stability of the functional structure. More generally, the structural rigidity and intricate networks of contacts in structured RNAs may allow them to evolve substantial structural specificity without explicit negative selection, even against closely related alternative structures.

SUBMITTER: Wan Y

PROVIDER: S-EPMC3227551 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Enhanced specificity against misfolding in a thermostable mutant of the Tetrahymena ribozyme.

Wan Yaqi Y Russell Rick R

Biochemistry 20110111 5

Structured RNAs encode native conformations that are more stable than the vast ensembles of alternative conformations, but how this specificity is evolved is incompletely understood. Here we show that a variant of the Tetrahymena group I intron ribozyme that was generated previously by in vitro selection for enhanced thermostability also displays modestly enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective press ...[more]

PMID: 21174447

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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

Enhanced specificity against misfolding in a thermostable mutant of the Tetrahymena ribozyme.

Publications

Enhanced specificity against misfolding in a thermostable mutant of the Tetrahymena ribozyme.

Similar Datasets

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