Role of Ion Valence in the Submillisecond Collapse and Folding of a Small RNA Domain

Abstract

Following the addition of ions to trigger folding, RNA molecules transition from rigid, extended states to a compact ensemble. Determining the time scale for this collapse provides important insights into electrostatic contributions to RNA folding; however it can be challenging to isolate the effects of purely non-specific collapse, e.g. relaxation due to backbone charge compensation, from the concurrent formation of some tertiary contacts. To solve this problem, we decoupled non-specific collapse from tertiary folding using a single point mutation to eliminate tertiary contacts in the small RNA subdomain known as tP5abc. Microfluidic mixing with microsecond time resolution and FRET detection provides insight into the ionic strength dependent transition from extended to compact ensembles. Differences in reaction rates are detected when folding is initiated by monovalent or divalent ions, consistent with equilibrium measurements illustrating the enhanced screening of divalent ions relative to monovalent ions at the same ionic strength. Ion-driven collapse is fast and a comparison of the collapse time of the wild type and mutant tP5abc suggests that site binding of Mg2+ occurs on submillisecond time scales.

Understanding the kinetic mechanism of RNA single base pair formation

“…3C. The U state contains unfolded clusters, such as M 21 , M 23 , and M 44 . In these clusters, the base flips out and both the base and the backbone (of G1) have nonnative conformations.…”

“…Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). However, due to the complexity of the RNA folding energy landscape (37)(38)(39)(40)(41)(42)(43)(44)(45)(46) and the limitations of experimental tools (47)(48)(49)(50)(51)(52)(53)(54)(55), many fundamental problems, ...…”

Predicting 3D Structure, Flexibility, and Stability of RNA Hairpins in Monovalent and Divalent Ion Solutions

“…4a and Fig. 4b shows that Na + and Mg 2+ induce a similar structural transition from a bent state at low ion concentrations to a coaxially stacked state at high salt, while Mg 2+ is much more efficient in inducing such structure transition due to the higher ionic charge (55)(56)(57)(58).…”

“…Combined with an implicit-salt/solvent force field and the Monte Carlo (MC) simulated annealing algorithm, the model can not only predict native-like 3D structures of small RNAs from their sequences at a high salt (e.g., 1M NaCl), but also give reliable predictions on the stability for RNA hairpins over a wide range of sequences and monovalent ion concentrations as compared with extensive experimental data (54). However, compared with monovalent ions (e.g., Na + ), divalent ions such as Mg 2+ can play a more special role in the stability and dynamics of RNA structures (55)(56)(57)(58)(59)(60). For example, Mg 2+ is about 1000 times more efficient in inducing the tertiary 3 structures folding of Tetrahymena thermophila ribozyme (45,49).…”

Formation of Tertiary Interactions during rRNA GTPase Center Folding

“…We assign this event to global electrostatic relaxation by nonspecific association of Mg 2+ ions. The phenomenon of rapid Mg 2+ -dependent (electrostatic) global collapse of large RNAs has been reported for several systems[30][31,32][33,34], even for RNAs that cannot form tertiary structure (e.g., the 303 nucleotide bI5core RNA [35]). For now, we will continue to refer to the very rapid fluorescence changes of GAC as electrostatic relaxation, where addition of Mg 2+ ions reduces the local charge density of the RNA and facilitates its exploration of local and global structures.…”