RNA Structural Modules Control the Rate and Pathway of RNA Folding and Assembly

Abstract

Structured RNAs fold through multiple pathways, but we have little understanding of the molecular features that dictate folding pathways and determine rates along a given pathway. Here, we asked whether folding of a complex RNA can be understood from its structural modules. In a two-piece version of the Tetrahymena group I ribozyme, the separated P5abc subdomain folds to local native secondary and tertiary structure in a linked transition and assembles with the ribozyme core via three tertiary contacts: a kissing loop (P14), a metal core-receptor interaction, and a tetraloop-receptor interaction, the first two of which are expected to depend on native P5abc structure from the local transition. Native gel, NMR, and chemical footprinting experiments showed that mutations that destabilize the native P5abc structure slowed assembly up to 100-fold, indicating that P5abc folds first and then assembles with the core by conformational selection. However, rate decreases beyond 100-fold were not observed because an alternative pathway becomes dominant, with nonnative P5abc binding the core and then undergoing an induced-fit rearrangement. P14 is formed in the rate-limiting step along the conformational selection pathway but after the rate-limiting step along the induced-fit pathway. Strikingly, the assembly rate along the conformational selection pathway resembles that of an isolated kissing loop similar to P14, and the rate along the induced-fit pathway resembles that of an isolated tetraloop-receptor interaction. Our results indicate substantial modularity in RNA folding and assembly and suggest that these processes can be understood in terms of underlying structural modules.

Keywords: P5abc; RNA folding; biomolecular interactions; group I intron; three-helix junction.

Fig 1

Folding and assembly of P5abc. (a), Secondary structure changes in P5abc induced by Mg²⁺ binding (blue). Point mutations designed to shift the equilibrium between these two structures are indicated with arrows. Mutations indicated in red stabilize the non-native secondary structure and the mutation in purple (G176A) stabilizes the native secondary structure. (b), The overall Mg²⁺-induced folding transition depicted with structures of the alternative and native P5abc structures based on NMR and X-ray crystallography, respectively [15, 32]. Nucleotides that change base pairing are colored blue as above, and site-bound Mg²⁺ ions are depicted as orange spheres [15, 65, 66]. (c), P5abc assembly with the E^ΔP5abc ribozyme core. The complex includes three long-range tertiary contacts: a tetraloop-tetraloop receptor interaction (TL/TLR, red), a metal core-metal core receptor interaction (MC/MCR, magenta), and a kissing loop formed by base pairing between L5c and L2 (P14, green).

Fig 2

P5abc assembly with the E^ΔP5abc ribozyme core. (a), Minimal model for coupled folding and assembly of P5abc with E^ΔP5abc by conformational selection. (b), Pulse-chase assay to measure assembly kinetics. E^ΔP5abc and ³²P-labeled P5abc (P5abc*) were separately incubated in Mg²⁺ solution to permit folding, and then they were mixed together to initiate assembly. After various times t, further association of P5abc* was blocked by adding excess unlabeled P5abc. The concentration of Mg²⁺ was also increased to 50 mM to prevent dissociation of bound P5abc*. (c), Progress curves for assembly of wild-type P5abc (black) and U167C P5abc (red) at 25 °C, 10 mM Mg²⁺. The E^ΔP5abc concentration was 10 nM (▽), 25 nM (□), and 100 nM (△) for reactions with wild-type P5abc, and 100 nM (▲), 250 nM (●), and 1 uM (◢) for reactions with U167C P5abc. (d), Dependence of the observed rate constant on E^ΔP5abc concentration. P5abc variants were: wild type (▽), G176A (●), U167C (△), U167C/G176A (▲), G174A/G176A (○), U177C/G176A (◇), and U167C/U177C/G176A (□). All P5abc mutants that include the G176A mutation are shown in purple. (e), The slowly assembling mutants shown on a scale that allows visualization of the concentration dependences. Wild-type P5abc is shown in black (▽) and mutants U167C (△, reproduced from panel d for comparison), G174A (□), U177C (◇), U167C/U177C (▲), and G174A/U177C (■) are red. The blue dashed line shows the behavior of U167C/U177C predicted from the conformational selection model in panel (a) with additive effects of the two mutations.

Fig 3

Equilibrium measurements of P5abc mutants by NMR. (a), 2D ¹H-¹⁵N SOFAST-HMQC spectra measured in the absence of Mg²⁺ (upper panels) and in the presence of 5 mM Mg²⁺ (lower panels; 2 mM Mg²⁺ was used for G176A to avoid severe signal deterioration). The U142 and U142* peaks are highlighted by black circles. (b), 1D imino SOFAST-HMQC spectra measured under the specified Mg²⁺ concentrations. The U142 and U142* peaks are highlighted by red labels. For all NMR experiments, RNA concentrations were 0.1 mM and spectra were collected at 10 °C.

Fig. 4

Evidence for an alternative assembly pathway. (a), Model for core assembly by the alternative (left) and native (right) P5abc structures. The L2 mutation A46C is expected to disrupt a base pair in the transition state for the native P5abc and therefore slow assembly (right), whereas this mutation is not expected to slow assembly with alternative P5abc. Nucleotides A46 and U168, which form this P14 base pair in the native conformation, are red, and P5c nucleotides that change secondary structure between the alternative and native structures are blue. (b), Assembly rate constants for wild-type P5abc and the indicated P5abc mutants with the wild-type E^ΔP5abc core (squares) or the E^ΔP5abc mutant A46C (triangles) at 10 mM MgCl₂ and 25 °C. Error bars representing the standard error from at least two determinations are present but not visible because they are smaller than the markers.

Fig. 5

Two pathways for RNA assembly. (a), The conformational selection pathway (blue), which dominates the flux for the wild-type P5abc and mutants that include the native-stabilizing G176A mutation. From the alternative conformation (P5abc_alt), P5abc rearranges to the native conformation (indicated as ‘local folding’ on the projection below the free energy profiles) and then assembles with the ribozyme core (yellow). This pathway dominates for these P5abc variants because the rate-limiting transition state (blue disc) is lower in free energy than that along the red, induced fit pathway (red disc). (b), The induced fit pathway (red), which dominates for P5abc mutants in which the alternative conformation is strongly stabilized relative to the native conformation. For these mutants, the maximal free energy of the rate-limiting step along the conformational selection pathway (blue disc) is increased sufficiently such that this pathway is disfavored relative to the induced fit pathway. After assembly, these P5abc mutants rearrange to the native conformation (local folding). In panels a and b, the cartoons in brackets depict proposed structural features of the rate-limiting transition states (P14 along the conformational selection pathway and the TL/TLR interaction along the induced fit pathway). (c), Dependence of the observed assembly rate constant and dominant pathway on the equilibrium for native P5abc formation. The dependences of rate constant on K_eq for each pathway are shown as dashed lines with colors corresponding to the labels and the black dashed line shows the observed rate constant predicted by the model shown in (a) and (b). The regime to the right of the vertical black line is occupied by the wild-type P5abc and the native-stabilizing mutant G176A. Here, the equilibrium favors the native P5abc structure relative to the alternative structure (K_eq > 1) and the observed rate constant for assembly with the ribozyme core (black dashed curve) reflects binding of native P5abc. As native P5abc folding becomes modestly unfavorable in double mutants that include G176A (0.01 < K_eq < 1, center regime), the assembly rate constant decreases and the conformational selection pathway remains dominant. Further stabilization of the alternative structure (e.g. U167C, left of the vertical red line) results in the conformational selection pathway becoming slower than the induced fit pathway (K_eq < 0.01, left regime), such that the induced fit pathway dominates the flux. (d), Rapid rearrangement of P5abc follows initial assembly along the induced fit pathway. The asterisk indicates that the value reflects the measured rate of dissociation for an isolated TL/TLR contact [42]. See the Discussion for further details.