Extended structures in RNA folding intermediates are due to nonnative interactions rather than electrostatic repulsion

Abstract

RNA folding occurs via a series of transitions between metastable intermediate states for Mg(2+) concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.

Figure 1. B. subtilis S-domain structure

A) Secondary structure presentation of the S-domain folding intermediate. The structure is composed of three helical domains (boxed) that meet in a core. B) Ribbon diagram of the structural model of the folding intermediate. C) Native structure of the S-domain (1nbs.pdb) which is much more compact.

Figure 2. SAXS data and reconstruction for B. subtilis S-domain and DTL mutant

A) Scattering data and P(r) at 37 °C are shown. I(0) values are normalized to the same value while P(r) distributions are unit normalized (∫P(r) dr = 1). B) Shown are three orthogonal views of the molecular envelopes derived from the scattering data along with the S-domain crystal structure and model of the intermediate. The intermediate remains extended in the background of 200 mM NaCl while the removal of the tetraloop interaction (ΔTL) renders the molecule unable to fold to the native state, remaining in an extended conformation similar to the wild-type intermediate. The scale for the reconstructions and models is the same to allow for direct comparison.

Figure 3. Characterization of the B. subtilis intermediate structure by LD simulation

A) All-atom LD simulations of the intermediate with an extended P9 region. Overlay of five different simulation trajectories at 2 ns. The overall conformation is globally very similar, but locally diverse. Simulation was performed using an S-domain with extended P9 helix to better visualize the global similarities and differences of the five runs. B) Close-up view of the intermediate core. Though variability in H-bonds exists, the main shape of the core and the whole molecule changes little.

Figure 4. Characterization of the B. subtilis C134 U core mutant by SAXS

A) Mg²⁺-dependent folding of the wild-type S-domain and C134U mutant monitored by CD spectroscopy. B) Scattering data and P(r) distributions for the mutant at low and high ionic conditions. I(0) values are normalized to the same value while P(r) distributions are unit normalized (∫P(r) dr = 1). C) Three orthogonal views of the molecular envelopes derived from the scattering data are shown. Under high salt conditions, the Rg of the C134U intermediate is smaller by 2 Å relative to its low salt counterpart as well as the wild-type intermediate at low and high salt conditions. The scale for two reconstructions is the same to allow for direct comparison.

Figure 5. SAXS analysis and reconstructions for the E. coli S-domain

A) Scattering data and P(r) distributions for the native state and the intermediate under low and high ionic conditions at 37 °C are shown. I(0) values are normalized to the same value while P(r) distributions are unit normalized (∫P(r) dr = 1). B) Three orthogonal views of the molecular envelopes derived from the scattering data are shown. The scale for the reconstructions is the same to allow for direct comparison.