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Journal of the American Chemical Society

RNA SHAPE chemistry reveals nonhierarchical interactions dominate equilibrium structural transitions in tRNA(Asp) transcripts.


PMID 15796531

Abstract

Current models assume that RNA folding is strongly hierarchical such that the base-paired secondary structure is more stable than and forms independently of the tertiary structure. This model has been difficult to test due to the experimental inability to interrogate the local environment at every nucleotide as a comprehensive function of the RNA folding state. Reaction of an RNA 2'-hydroxyl group with N-methylisatoic anhydride to form a nucleotide 2'-ester is governed by the extent to which the nucleotide forms base pairing or tertiary interactions. Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is shown to be an RNA analogue of the protein hydrogen exchange experiment. Single nucleotide resolution SHAPE analysis emphasizes a complexity for the unfolding of tRNA(Asp) transcripts that is not anticipated by current models for RNA folding. We quantify six well-defined transitions for tRNA(Asp) transcripts between 35 and >75 degrees C, including asymmetric unfolding of the two strands in a single helix, multistep loss of tertiary interactions, and a multihelix conformational shift. The three lowest temperature transitions each involve coupled interactions between the secondary and tertiary structure. Thus, even for this simple RNA, multiple nonhierarchical and complex interactions dominate the equilibrium transitions most accessible from the native state.

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