Taming free energy landscapes with RNA chaperones

Abstract

Many non-coding RNAs fold into complex three-dimensional structures, yet the self-assembly of RNA structure is hampered by mispairing, weak tertiary interactions, electrostatic barriers, and the frequent requirement that the 5' and 3' ends of the transcript interact. This rugged free energy landscape for RNA folding means that some RNA molecules in a population rapidly form their native structure, while many others become kinetically trapped in misfolded conformations. Transient binding of RNA chaperone proteins destabilize misfolded intermediates and lower the transition states between conformations, producing a smoother landscape that increases the rate of folding and the probability that a molecule will find the native structure. DEAD-box proteins couple the chemical potential of ATP hydrolysis with repetitive cycles of RNA binding and release, expanding the range of conditions under which they can refold RNA structures.

Figure 1

RNA folding pathways and refolding by chaperones. In the absence of chaperones (top), the initial collapse transition produces an ensemble of compact intermediates (I_C) that rearrange to the native structure (N). Because the unfolded RNA contains a mixture of structures, including non-native base pairs (yellow), different subpopulations (U¹, U², U³) fold through different pathways (reviewed in ref. 134). Non-specific collapse transitions lead to kinetically trapped misfolded intermediates (I_mis). Chaperone proteins (C) bind and destabilize RNA folding intermediates, releasing partially unfolded RNAs that can fold again. The structure of the RNA immediately after chaperone release is unknown (question marks).

Figure 2

Free energy landscapes for RNA folding. When conditions favor the unfolded state (e.g., low salt), RNA interactions are weak and molecules can adopt many different structures (top). When the RNA population is jumped to native conditions (e.g., Mg²⁺), RNA interactions are stable and the molecules seek out low free energy structures such as the native state (N) (bottom). Free energy landscapes for RNA folding are rough, because non-native structures are stable, corresponding to local minima on the energy landscape (I_mis).,, A fraction of the population Φ takes a direct route to N and folds rapidly. Molecules that are initially trapped in misfolded structures must cross high transition states (red arrow), and thus fold slowly.

Figure 3

Thermodynamics of RNA unfolding by non-enzymatic chaperones. (A) Thermodynamic cycle for chaperone (C) binding to unfolded (U) and folded (F) RNA, in which the folded RNA can be an intermediate (I) or native (N) form. The difference in the free energy of chaperone binding to U and F (ΔG_b^F − ΔG_b^U) is equal to the perturbation of the folding free energy by the chaperone (ΔG_f − ΔG_f^C). (B) Chaperones that interact tightly with unfolded RNA lower the free energy of U relative to I and N. If the unfolded RNA-chaperone complex (U•C) becomes too stable, the energy gap ΔG_net between the U•C complex and N is small, and folding is less favorable overall.

Figure 4

Counterion charge density and RNA folding dynamics. (A) The valence and size or charge density, of counterions influences the stability of RNA structure. Small, multivalent ions (Mg²⁺, left) stabilize RNA tertiary structures more than larger ions (polyamines, middle). Basic polypeptides in RNA chaperones may destabilize RNA structures by lowering the density of positive charge around the RNA (right). (B) Chaperones can smooth free energy landscapes for RNA folding by destabilizing I states (green arrow) and stabilizing transition state ensembles (TSE; blue arrow). As the landscape becomes smoother, the transition state for collapse moves toward N, increasing the probability Φ an RNA will fold correctly and bypass non-native I's.