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.pathway ͉ ribozyme ͉ salt ͉ assembly ͉ topology M ost of the studied RNA folding pathways are populated with multiple kinetic intermediates (1-9) whose prevalence suggests their important role in the regulation of RNA function (10-14). The structural properties of folding intermediates and the factors that influence their population and lifetime are incompletely understood. Among the known factors are intermediate stability, electrostatic environment, nativestate topology, and the initial conformational ensemble (15). RNA's negative charge results in counterion concentration strongly influencing the folding landscape and the relative stability of kinetic intermediates (16)(17)(18)(19)(20). The native-state topology of structured RNA consisting of Watson-Crick duplexes connected by semiflexible junctions and specific long-range tertiary contacts constrains the biopolymer's conformational degrees of freedom (21-23). The ensemble of macromolecular conformations present at the initial state directs the RNA commitment to a particular folding pathway (16): a compact ensemble with significant tertiary structure may be more favorable to conformational search but can also yield barriers that impede resolution of misfolded molecules. The distinct roles of each of these influences in the determination of the structure, population, and lifetime of RNA folding intermediates have yet to be established.In this study we investigate the interplay among electrostatics, initial conformation, and macromolecular stability in RNA folding. We analyze the solution condition dependence of the intermediate structures and molecular flux through them on the Mg 2ϩ -mediated folding of the L-21 Tetrahymena thermophila group I intron and a mutant of reduced stability by varying the background monovalent cation type and co...

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

“…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. Structural-kinetic folding models, by providing insight into the dominant folding pathways, structural features of the folding intermediates, and their solution lifetimes Shcherbakova and Brenowitz 2008), have helped elucidate the contributions of both physical and intrinsic factors that influence RNA folding mechanisms (Laederach et al 2007). …”

3 Importance of Diffuse Metal Ion Binding to RNA

“…The stabilization of the nonspecific collapsed state slows down the (productive) folding to the final native state which requires the formation and stabilization of specific native contacts [49,67]. In addition, a recent experiment showed that, depending on the ionic condition of the solution, the kinetic partitioning of the folding flux can be dependent on the initial conformational ensemble [68].…”

“…By lowering the electrostatic barrier for the kinetics, ions can promote the kinetic process of RNA folding and control the folding rate and pathways. There have been tremendous experimental efforts on the role of metal ions in RNA (and DNA) folding kinetics (see Table 4) at both secondary and tertiary structural levels [20,[61][62][63][64][65][66][67][68].…”