Kinetic redistribution of native and misfolded RNAs by a DEAD-box chaperone

Abstract

DExD/H-box proteins are ubiquitously involved in RNA-mediated processes and use ATP to accelerate conformational changes in RNA. However, their mechanisms of action, and what determines which RNA species are targeted, are not well understood. Here we show that the DExD/H-box protein CYT-19, a general RNA chaperone, mediates ATP-dependent unfolding of both the native conformation and a long-lived misfolded conformation of a group I catalytic RNA with efficiencies that depend on the stabilities of the RNA species but not on specific structural features. CYT-19 then allows the RNA to refold, changing the distribution from equilibrium to kinetic control. Because misfolding is favoured kinetically, conditions that allow unfolding of the native RNA yield large increases in the population of misfolded species. Our results suggest that DExD/H-box proteins act with sufficient breadth and efficiency to allow structured RNAs to populate a wider range of conformations than would be present at equilibrium. Thus, RNAs may face selective pressure to stabilize their active conformations relative to inactive ones to avoid significant redistribution by DExD/H-box proteins. Conversely, RNAs whose functions depend on forming multiple conformations may rely on DExD/H-box proteins to increase the populations of less stable conformations, thereby increasing their overall efficiencies.

Figure 1

Unfolding of native and misfolded Tetrahymena ribozyme. a, Reaction scheme. b, Substrate cleavage after incubation with 1 mM Mg²⁺, 2 μM CYT-19 and 2 mM ATP-Mg²⁺ for 0.25 (orange), 0.67 (red), 1 (cyan), 2.5 (magenta), 9.5 (blue), or 22 min (dark green). (Light green), no CYT-19. c, Native ribozyme unfolding (1 mM Mg²⁺). CYT-19 was 1 μM (∇), 2 μM (solid colored circles), or 3 μM without (σ) or with 2 mM ATP-Mg²⁺ (∆). Colored circles show burst amplitudes from corresponding curves (panel b). ○, no CYT-19; ●, 2 μM CYT-19, 2 mM ATP-Mg²⁺, 5 mM Mg²⁺. d, Rate constant (○) and steady-state value (∇) vs CYT-19 concentration. e, Approach to steady state from native (○) or misfolded (∇) ribozyme with 1.2 μM (blue) or 2 μM (red) CYT-19. f, Refolding to the native state (∇) after unfolding by CYT-19 (◇) and inactivation by proteinase K. ○, no CYT-19.

Figure 2

Secondary structure, long-range tertiary contacts, and mutations of the Tetrahymena ribozyme. The five long-range tertiary contacts are indicated with red arrows. In the P5abc deletion variant (E^ΔP5abc), the region shown in yellow is deleted and nucleotides 126 and 196 are directly connected (thick gray line above yellow region). In the P5a mutant, nucleotides 183-188 (shaded cyan) are each changed to uridine. This mutation disrupts the tertiary contact indicated by the black ‘X’.

Figure 3

Unfolding of destabilized ribozyme variants. a, P5a variant with 0.8 μM CYT-19 (◇). Upon CYT-19 inactivation, native ribozyme accumulated (∆, 0.056 min^-1) with the same rate constant within error as for refolding of the misfolded ribozyme (○, 0.032 min^-1). b, Approach to steady state for native (○) or misfolded (∇) P5a variant with 0.5 μM (blue) or 1 μM (red) CYT-19. Curves depict kinetic simulations using experimentally-derived values (see Supplementary Fig. 6), not fits to the data. c, Approach to steady state for E^ΔP5abc ribozyme. Curves and symbols as in panel b except red (1.2 μM CYT-19). d, Unfolding of native (◇) and misfolded (○) E^ΔP5abc ribozyme. All reactions were 25 °C, pH 7.0, 5 mM Mg²⁺.

Figure 4

Model for chaperone activity. CYT-19 generates ribozyme intermediates (I) and then allows them to fold again, thus increasing the population of less stable intermediates that are kinetically favored. Values are for the P5a variant, normalized by native ribozyme unfolding (red; (1) indicates 5 × 10⁵ M^-1 min^-1, Supplementary Fig. 3), or ‘direct’ native state formation (black; (1) indicates 0.4 min^-1, Supplementary Fig. 5). The wild-type ribozyme behaves similarly at low Mg²⁺ concentration, but at higher Mg²⁺ concentration the native species is sufficiently stable that CYT-19 unfolds it poorly and therefore can accelerate its formation from the kinetically-trapped misfolded species.

Scheme 1