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Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding.

ABSTRACT: We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved "Fast Fenton" hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel pathways leading the RNA to its native form upon its Mg(2+)-induced folding are observed. The structures of the intermediates populating the pathways are not affected by variation of the concentration and type of background monovalent ions (electrostatic environment) but are altered by a mutation that destabilizes one domain of the ribozyme. Experiments starting from different conformational ensembles but folding under identical conditions show that whereas the electrostatic environment modulates molecular flux through different pathways, the initial conformational ensemble determines the partitioning of the flux. This study showcases a robust approach for the development of kinetic models from collections of local structural probes.

SUBMITTER: Laederach A

PROVIDER: S-EPMC1855354 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding.

Laederach Alain A Shcherbakova Inna I Jonikas Magdalena A MA Altman Russ B RB Brenowitz Michael M

Proceedings of the National Academy of Sciences of the United States of America 20070416 17

We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved "Fast Fenton" hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel ...[more]

PMID: 17438287

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Project description:When a ribonucleic acid (RNA) molecule folds, it often does not adopt a single, well-defined conformation. The folding energy landscape of an RNA is highly dependent on its nucleotide sequence and molecular environment. Cellular molecules sometimes alter the energy landscape, thereby changing the ensemble of likely low-energy conformations. The effects of these energy landscape changes on the conformational ensemble are particularly challenging to visualize for large RNAs. We have created a robust approach for visualizing the conformational ensemble of RNAs that is well suited for in vitro versus in vivo comparisons. Our method creates a stable map of conformational space for a given RNA sequence. We first identify single point mutations in the RNA that maximally sample suboptimal conformational space based on the ensemble's partition function. Then, we cluster these diverse ensembles to identify the most diverse partition functions for Boltzmann stochastic sampling. By using, to our knowledge, a novel nestedness distance metric, we iteratively add mutant suboptimal ensembles to converge on a stable 2D map of conformational space. We then compute the selective 2' hydroxyl acylation by primer extension (SHAPE)-directed ensemble for the RNA folding under different conditions, and we project these ensembles on the map to visualize. To validate our approach, we established a conformational map of the Vibrio vulnificus add adenine riboswitch that reveals five classes of structures. In the presence of adenine, projection of the SHAPE-directed sampling correctly identified the on-conformation; without the ligand, only off-conformations were visualized. We also collected the whole-transcript in vitro and in vivo SHAPE-MaP for human β-actin messenger RNA that revealed similar global folds in both conditions. Nonetheless, a comparison of in vitro and in vivo data revealed that specific regions exhibited significantly different SHAPE-MaP profiles indicative of structural rearrangements, including rearrangement consistent with binding of the zipcode protein in a region distal to the stop codon.

Dataset Information

Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding.

Publications

Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding.

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