Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme

Abstract

Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are "brittle," breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

Fig. 1

(A) Secondary structure of the L-21 ribozyme. The two main domains, P4-P6 and P3-P8, are boxed; a light blue box indicates the catalytic core. Gray lines label sequences targeted by complementary DNA oligonucleotides; dashed lines are tertiary contacts (T) and base-paired regions (P); “M” labels are site-directed mutations (M1, A to U at site 186; M2, C to G at 260; M3, UGC to ACG at 348 to 350; M4, U to A at 273; M5, cyclic permutation at 148); cc is the catalytic core. The letters a, b, c, d, e, f, g, and h indicate the proposed positions of the kinetic barriers; the evidence for these assignments is presented throughout the text. (B) Representative unfolding (black) and refolding (pink) force/extension curves of the L-21 RNA displaying six unfolding events (rips). The RNA is attached by RNA · DNA handles to polystyrene beads, which are manipulated by the laser tweezers. The DNA components of the handles were prepared by polymerase chain reaction from the pBR322 plasmid. The handles do not appear to affect the functional integrity of the ribozyme, as verified by standard bulk catalysis assays (). Experiments were done at 298 ± 2 K in 10 mM Tris (pH = 7), 250 mM NaCl, and 10 mM MgCl₂ unless otherwise noted. Our first goal was to correlate the rips to the unfolding of domains and subdomains in (A). Letters and arrows correspond to the positions we assigned to the kinetic barriers as described in the text. The unfolding curve chosen here does not display barriers d and g, indicated by the dashed arrows.

Fig. 2

(A) Force/extension curves for the P4-P6 domain: black, unfolding curve; pink, refolding curve. The solid lines are WLC curves for double-stranded RNA · DNA handles and an increasing number of RNA nucleotides as the unfolding progresses. The unfolding of the subdomains releases 50 nt ( f, red, P4-P6 helices), then 30 nt (g, black, P5 helix), then 70 nt (h, blue, P5abc three-helix junction). The color code of the WLC curves corresponds to the motifs broken; it is identical to the motif color code in Fig. 1A. Inset, unfolding curves of P4-P6 in the presence of 10 μM antisense DNA oligonucleotide I (Fig. 1A), preventing T3 and disrupting barrier f. (B) Mechanical unfolding map of the P4-P6 domain. Four different unfolding trajectories were detected at our experimental resolution. F, folded; U, unfolded. I, intermediates: I₁, P6 unfolded; I₂, P6-P4 unfolded; I₃, P6-P4-P5 unfolded; I₄, P6-P4-P5-P5a unfolded. Sample force/extension curves of the different trajectories are shown in the bottom panel. Trajectory C is observed most frequently. P4-P6 colors are as in Fig. 1A. (C) Histogram of rips detected in 732 unfolding curves of P4-P6: position (5-nt bin), mean force (pN), and frequency of the rips.

Fig. 3

(A) Unfolding force/extension curves of L-21ΔP9P2. Barriers f, g, and h are identical to those in Fig. 2A (P4-P6). The new green WLC curve (e) describes the barrier for unfolding of the newly added domain, P3-P8. Inset, unfolding curves of L-21ΔP2P9 in the presence of 10 μM antisense DNA oligonucleotide preventing catalytic core tertiary interactions (Fig. 1A, II). (B) Unfolding force/extension curve of L-21ΔP2. Characteristic barriers for the unfolding of L-21ΔP2P9 (e to h) are clearly identified. The new set of WLC curves (violet, brown, and turquoise) displays three new kinetic barriers (a, b, and c) present during the unfolding of the P9 extension. Barrier a is associated with P9.2; barrier b, with P9.1-P9.1a; and barrier c, with P9.

Fig. 4

(A) Blue curves, superposition of 32 L-21 unfolding curves displaying six of the eight kinetic barriers detected in this study. The new orange WLC curve (d) describes the barrier for unfolding of the newly added P2 extension. The kinetic barriers can be clustered as proximal to the beginning of unfolding (a to d, P9 and P2 extensions), intermediate (e to g, catalytic core), and distal (h, P5abc extension). Curves were smoothed with a Gaussian kernel. Experiments done to investigate the nature of the folded state are described in () (fig. S6). (B) Histogram of the rips as a function of their position for 110 curves. Letters indicate the positions of the barriers. As seen in (A), the rip corresponding to barrier a is very frequent but has a different slope than the other rips. Our rip detection algorithm was based on slope differences and leaves rip a poorly detected and poorly represented in (B).