Multistage collapse of a bacterial ribozyme observed by time-resolved small-angle X-ray scattering

Abstract

Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg(2+) to the Azoarcus group I ribozyme up to 80% of chains form compact structures in less than 1 ms. In 1 mM Mg(2+), the collapse transition produces extended structures that slowly approach the folded state, while > or = 1.5 mM Mg(2+) leads to an ensemble of random coils that fold with multistage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg(2+)-dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg(2+)-dependent charge neutralization and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.

Figure 1. Time-resolved SAXS of Azoarcus ribozyme folding

(A) Schematic view of stopped-flow mixer (SFM400). Syringes were loaded with unfolded RNA (1 mg/mL after mixing) in 20 mM Tris-HCl and folding buffer containing MgCl₂. The dead time (~0.6 ms) was minimized by a high flow rate and short distance from the small-volume mixer to the observation point. (B) Kratky plots of real-time folding data in 1.5 mM MgCl₂. Curves at 5 s (green) were in 5 mM MgCl₂. For time-resolved measurements (≤ 200 ms), 20 identical 1 ms datasets were averaged. Scattering data up to 5 s were acquired for 50 ms and averaged over 4 shots. For unfolded RNA in 20 mM Tris-HCl (orange) and folded RNA in 5 mM MgCl₂ (black) at equilibrium, data were collected for 1.6 s (4 times).

Figure 2. Structure of early folding ensembles in Mg²⁺

Time-dependent exponents, ν, in different MgCl₂ were determined from linear fits to log [I(Q) ~ Q^−ν] vs log Q for 0.05 < Q < 0.1 Å⁻¹ (see Fig. S3). Error bars are from the statistics of the fit. Dashed line corresponds to worm-like chain (ν = 1); Dotted line corresponds to random coil-like chain (ν = 2). For an ideal sphere, ν = 4. The circled symbols represent ν values at equilibrium.

Figure 3. Multi-stage collapse kinetics

Decrease in R_g over time from the unfolded state (60.5 Å) to the folded state (31 Å) during refolding in 5 mM MgCl₂. Error bars represent the uncertainty in R_g from the data inversion. Solid line is the best fit to a triple exponential decay function (τ₁ ≤ 0.2 ms, τ₂ = 20 ms, τ₃ = 170 s; see Table S1). Blue bar, 5-20 ms time window in which tertiary interactions are detected by hydroxyl radical footprinting . Inset: Pair distance distribution function, P(r), for RNA folding in 5 mM MgCl₂ at the times shown.

Figure 4. Mg²⁺ determines partitioning of collapse kinetics

(A) Fraction of unfolded Azoarcus ribozyme, Φ_U(t), refolded in 1, 1.5, 2, 5 and 10 mM MgCl₂. Solid lines represent the best fit to Eq. 1; fit parameters are in Table S1. See Fig. S4A,B for further data. (B) Partitioning of RNA population into burst (P₁; blue) and second phase (P₂; red) within 200 ms. 1-Φ_U,E is the fraction of folded RNA at equilibrium. The difference between 1-Φ_U,E (orange) and P₁ + P₂ (green) represents long-lived misfolded RNAs. Error bars are from the uncertainty of fits in (A).

Figure 5. Urea increases the fraction of rapidly compacting RNA

Time-dependent folding of 3 M urea-relaxed ribozyme compared with urea-free ribozyme. (A) Fraction unfolded Azoarcus ribozyme as in Fig. 4A. Solid lines, no urea; dashed lines, with 3 M urea. Colors denote conditions of isostability determined from equilibrium titrations (Fig. S1). See Fig. S4C for further data. (B) Partition factors for first two phases (P₁ and P₂) with urea (red) and without urea (blue). The Mg²⁺ concentration axis for urea-free reactions (top) is shifted by a factor of 1.4 relative to the lower x axis.

Figure 6

Folding nucleation and collapse of the Azoarcus ribozyme. Parallel folding from subpopulations of the unfolded state, U¹, U², and U³, leads to heterogeneous folding kinetics. Direct folding to the native state (U¹) is favored by higher Mg²⁺ concentration or by partially denaturing amounts of urea. U² is populated only when Mg²⁺ concentration is higher than the midpoint of the equilibrium folding transition (0.88 mM) and has a cusp at 1.5 - 2 mM Mg²⁺. Left and middle cartoons represent hypothetical structures in the unfolded and non-native relaxed coil ensembles. Average persistence lengths (l_p) are from fits to P(r) as previously described .