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Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function.

ABSTRACT: The Tetrahymena ribozyme derived from the self-splicing group I intron binds a 5'-splice site analog (S) and guanosine (G), catalyzing their conversion to a 5'-exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10(2) to 10(3) x s(-1)) and prior structural data suggest local rather than global rearrangements, raising the possibility that folding of this and perhaps other large RNAs is not fully cooperative. Guanosine binding is accelerated by addition of residues that form helices, referred to as P9.0 and P10, immediately 5' and 3' to the guanosine. These rate enhancements provide evidence for binding intermediates that have the adjacent helices formed before accommodation of guanosine into its binding site. Because the ability to form the P9.0 and P10 helices distinguishes the guanosine at the correct 3'-splice site from other guanosine residues, the faster binding of the correct guanosine can enhance specificity of 3'-splice site selection. Thus, paradoxically, the absence of a preformed binding site and the resulting slow guanosine binding can contribute to splicing specificity by providing an opportunity for the adjacent helices to increase the rate of binding of the guanosine specifying the 3'-splice site.

SUBMITTER: Karbstein K

PROVIDER: S-EPMC151335 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature

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Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function.

Karbstein Katrin K Herschlag Daniel D

Proceedings of the National Academy of Sciences of the United States of America 20030218 5

The Tetrahymena ribozyme derived from the self-splicing group I intron binds a 5'-splice site analog (S) and guanosine (G), catalyzing their conversion to a 5'-exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10(2) to 10(3) x s(- ...[more]

PMID: 12591943

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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

Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function.

Publications

Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function.

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