Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting

Abstract

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.

Figure 1

(a) The mIAP gag-pro frameshift site (Fehrmann et al., 1997). The slip site heptamer is enclosed in a box and the pseudoknot structural elements are annotated. The structure of this pseudoknot has yet to be determined. The adenosine at the junction (A15), shown unpaired in this model, may be paired with the final uridine in loop 2 (U31) and it is not clear which of the two adenosine bases (A35 and A36) in stem 2 is paired with U11. (b) Mutations incorporated in the mIAP pseudoknot sequence. The double mutant, ΔA35A15 RNA, is indicated by the open circles.

Figure 2

(a) The sequences of the mIAP pseudoknot stem 1 hairpin RNAs. (b) Melting profiles of the wild-type mIAP pseudoknot and two stem 1 hairpins at 50 mM KCl in the absence of exogenously added Mg²⁺. On the left, the composite fits are superimposed on the experimental data. On the right, the individual transitions of the composite fit are shown for clarity. Black (•) and gray-scale (+) correspond to ∂A/∂T monitored at 260 nm and 280 nm, respectively.

Figure 3

Optical and calorimetric melting profiles for (a) the mIAP pseudoknot RNA and (b) the deletion mutant, ΔA35 RNA. The composite fit is superimposed on the calorimetry data ( •) on the left, with the individual transitions shown for clarity on the right with the composite fit shown with a broken line. Optical data are represented as described previously with black ( •) and gray-scale (+) corresponding to ∂A/∂T monitored at 260 nm and 280 nm, respectively.

Figure 4

Melting profiles of the wild-type mIAP pseudoknot and stem assignment mutant RNAs at 50 mM KCl in the absence of exogenously added Mg²⁺. On the left, the composite fits are superimposed on the experimental data. On the right, the individual transitions of the composite fit are shown for clarity. Black ( •) and gray-scale (+) correspond to ∂A/∂T monitored at 260 and 280 nm, respectively.

Figure 5

The proposed equilibrium unfolding pathway of the mIAP pseudoknot based on a multiple, interacting, sequential two-state transition analysis of the optical and calorimetric unfolding data.

Figure 6

Effect of [Mg²⁺] on the t_m for the first two unfolding transitions in the wild-type mIAP pseudoknot. The dependence of 1/t_m on [Mg²⁺] for the first transition (F→S1+J) is shown at the top and for the second transition (S1+J→I) at the bottom (seeFigure 5). The continuous line represents the fit of the experimental data to a non-specific binding model (Nixon & Giedroc, 1998).

Figure 7

Melting profiles of the wild-type mIAP pseudoknot and unpaired adenosine deletion mutant RNAs at 50 mM KCl in the absence of exogenously added Mg²⁺. On the left, the composite fits are superimposed on the experimental data. On the right, the individual transitions of the composite fit are shown for clarity. Black (•) and gray-scale (+) correspond to ∂A/∂T monitored at 260 nm and 280 nm, respectively.

Figure 8

Urea dependence of the optical melting profiles for the mIAP pseudoknot and he deletion mutant ΔA15 RNAs in 50 mM KCl. (a) Wild-type mIAP in the left column and (b) ΔA15 in the right column at 0, 20, 40, and 50 % (w/v) urea. Black ( •) and gray-scale (+) correspond to ∂A/∂T monitored at 260 and 280 nm, respectively with the composite fits superimposed on the experimental data.

Figure 9

Melting profiles of the wild-type mIAP pseudoknot compared to the loop 2 substitution mutant, L2U RNA. On the left, the composite fits are superimposed on the experimental data. On the right, the individual transitions of the composite fit are shown for clarity. Black ( •) and gray-scale (+) correspond to ∂A/∂T monitored at 260 and 280 nm, respectively. The composite fit is superimposed on the L2U calorimetry data ( •) on the left, with the individual transitions shown for clarity on the right with the composite fit shown with a broken line.