Local Kinetic Measures of Macromolecular Structure Reveal Partitioning among Multiple Parallel Pathways from the Earliest Steps in the Folding of a Large RNA Molecule

Abstract

At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical (()OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg(2+) and Na(+)-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg(2+)-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na(+)-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…Parallel advances have been made with a new and powerful footprinting approach called "SHAPE", which shows protection data for residues in secondary and tertiary structures [112][113][114]. New kinetic modeling approaches have greatly simplified and systematized the development of kinetic models from large sets of footprinting data; such an approach has been recently applied to characterize the folding pathways of wild type and mutant Tetrahymena group I intron [115,116].…”

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

“…A well-established tenet in RNA folding is that even subtle perturbations in the stability of the native state alter the dominant folding pathways (Shcherbakova and Brenowitz 2005;Laederach et al 2006). Therefore, in order to address the relationship between topologically conserved tertiary interactions and folding pathways in RNA, we adopted a complementary quantitative approach that critically compares structural-kinetic folding models resolved from timeresolved hydroxyl radical ( d OH) footprinting experiments on structurally homologous RNA molecules.…”

Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme

“…Several lines of evidence have indicated that one of the peripheral elements, termed P5abc, plays a vital role in folding. Time-resolved oligonucleotide hybridization and footprinting studies showed that tertiary folding begins with P5abc, followed closely by the rest of the P4-P6 domain (2,3,17,18) (Fig. 1).…”

“…1). The rest of the ribozyme then acquires stable structure through a series of intermediates (2,3,18), while P5abc and the entire P4-P6 domain retain structure in each subsequent intermediate. In addition to being the first subdomain to form, P5abc is the most stable part of the ribozyme, remaining ordered at the lowest Mg 2+ concentration and the highest temperature (19,20).…”