The rate-limiting step in the folding of a large ribozyme without kinetic traps

Abstract

A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions.

Figure 1

(A) The B. subtilis C-domain (47). A fluorescein is covalently attached to the 5′ end that allows monitoring the folding kinetics by fluorescence spectroscopy. (B) Folding kinetics at 10°C starting from either the U_aq state (20 mM Tris⋅HCl, no Me²⁺) or the U_urea state (20 mM Tris⋅HCl, 8 M urea, 1 mM EDTA, no Me²⁺). Folding is monitored by absorbance from 0.002 to 0.2 s, and the dead-time change in signal is indicated with dashed arrows. Folding is monitored by fluorescence between 0.2 to 360 s. The fluorescence signal is fit with a two-exponential function. The slow phase has the same rate as that obtained by catalytic activity (open circles, right axis) and directly reports the rate-limiting step. The fast phase corresponds to the signal change of the I_eq-to-I transition and is ≈6 times faster than the slow phase.

Figure 2

(A) Metal-ion chevron analysis using fluorescence spectroscopy (Upper). In the chevron analysis, rate saturation at the high or low [Me²⁺] indicates the formation of a folding and unfolding intermediate, I and I, respectively. [Me²⁺] at the vertex of the chevron is approximately the same as the midpoint of the equilibrium I_eq-to-N transition measured independently (Lower). (B) Folding kinetics of C-domain from I_eq at 10 mM MgCl₂, 10°C, monitored by SAXS. The percentage of compaction is calculated from radius gyration. This result indicates that I is highly compact and forms quickly.

Scheme 1

Figure 3

(A) Temperature dependence of the folding rate. (B) Correlation between the activation enthalpy for folding and the dehydration of the metal ions measured by NMR (36).

Figure 4

Summary of C-domain folding. The rate-limiting step is shown in red. The folding half-lives at 10°C are shown, in seconds, above the arrows. The starting point is the U_urea state (C domain in 8 M urea and 1 mM EDTA), which has little RNA structure. I_eq contains the native amount of secondary structure and some tertiary structure. I contains >98% of the native structure. In less than 1 ms, the majority of the conformational search occurs with the formation of I_eq. All folding half-lives are measured in this work. Percent compaction is calculated from the data shown in Fig. 2B and from our previous work (31). Percent m-value change is calculated from published data (15).