Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for −1 ribosomal frameshifting stimulation

Figures

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  Figure 1. Pseudoknot structures and optical tweezers experimental setup. (a) Secondary structure of pseudoknot SF206 and mutants. The slippery sequence (G GGA AAC) is indicated by a vertical black line. The DNA strands (highlighted in dark gray) are complementary to the mRNA (highlighted in light gray) forming the handles A and B for the pulling experiments. (b) Experimental setup of single-molecule force spectroscopy using Minitweezers. The NMR structure18 is shown with the same color coding as panel a. The drawing is not to scale. (c) Structures of the three consecutive minor-groove base triples A28∙C6-G14 (cyan), U27∙C5-G15 (green), and A26∙G4-C16 (magenta) found in SF206.

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  Figure 2. Ensemble thermal melting studies reveal no correlation between thermal stability and frameshifting efficiency. Error bars for Tm,1, Tm,2 and frameshifting efficiency represent standard deviations. (a) Normalized UV melting trajectories of pseudoknot SF206 in the buffers with 1 M (red), 200 mM (green) and 0 mM (blue) NaCl, respectively. Solid lines represent the heating curve and dashed lines represent the cooling curve. (b) Normalized UV melting trajectories of isolated stem 1 hairpin (containing residues from 1–19) in the buffers with 1 M (red), 200 mM (green) and 0 mM (blue) NaCl, respectively. Solid lines represent the heating curve and dashed lines represent the cooling curve. (c) Normalized UV melting trajectories of pseudoknots and control hairpin in the buffer with 0 mM NaCl. SF348 shows only the melting of stem 1 suggesting that the pseudoknot structure for SF348 does not form at this condition. (d) No correlation is observed between Tm,2 in 0 mM NaCl and in vitro frameshifting efficiency previously measured19. The blue solid line and the black dashed lines represent the linear fitting curves for data points with and without SF209 and SF211 included, respectively. (e)…

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  Figure 3. Native PAGE results for RNA oligonucleotides (consisting of residues from 1 to 34 for the pseudoknots and residues 1–19 for the hairpin containing stem 1). The same constructs were used for the thermal melting studies. The stem 1-loop 2 minor-groove base triple formation is maintained in SF206, SF209, and SF211. The mutants SF217 and SF220 have a single base mutation of a residue in loop 2 involved in base triple interactions.

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  Figure 4. Representative single-mechanical (un)folding traces. (a,b) Representative force-ramp pulling traces observed for SF209 with a typical one-step unfolding pathway (a) and a rarely observed two-step unfolding pathway (b). The curves are smoothed to 100 Hz. Red curves are unfolding traces and black curves are refolding traces. The unfolding force is indicated by a horizontal grey arrow. (c,d) Representative force-jump traces for the one-step unfolding and two-step refolding of SF206 at 27.0 and 15.0 pN, respectively. The 1000 Hz data (cyan) were subsampled to 20 Hz (blue). The dashed lines indicate the Gaussian distribution fitting results. Extensions were binned to 1 nm, and forces were binned to 0.05 pN.

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  Figure 5. Comparison between thermal and mechanical (un)folding pathways and scheme of ribosomal −1 frameshifting. (a) Proposed thermal unfolding and folding pathways (see Fig. 2). (b) Proposed single-molecule mechanical unfolding and folding pathways. The stabilizing effect of coaxial stacking between stem 1 (red) and stem 2 (blue), and the base triples formed between stem 1 and loop 2 (green) on the component stems (stem 1 and stem 2) is revealed by analyzing the one-step mechanical unfolding forces. (c) Scheme of ribosomal frameshifting from 0 frame to −1 frame. The mRNA slippery sequence and single-stranded spacer within the ribosome are shown in yellow and gray, respectively. Stem 1-loop 2 base triple formation in the mRNA pseudoknot may enhance the local stability of stem 1 and result in enhanced frameshifting. The tRNAs and nascent peptide are shown in magenta and brown, respecitvely. The drawing is not to scale.

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  Figure 6. SMD simulation results for SF206. (a) An averaged curve of a total of 10 independent pulling simulations. The green error bars are 95% confidence intervals. The end-to-end distance is defined as the distance between the O3′ atom of the 3′ terminal residue A and the O5′ atom of the 5′ terminal residue G. The vertical dotted lines in panels a and b correspond to the highest peak. (b) Averaged number of hydrogen bonds formed as a function of extension based on the 10 independent simulations. (c–g) Structural snapshots corresponding to the positions in panel a. The snapshots are taken from a representative simulation.

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  Figure 7. Relationship between mechanical unfolding and frameshifting. (a) In vitro frameshifting efficiency19 versus mean unfolding force. (b) Unfolding kinetics of SF206 extracted from force distributions using Dudko’s method52. Bell’s model is used to fit the force-dependent unfolding rates to extract rate constants at different forces (k) and unfolding transition state positions (X≠). (c–e) In vitro frameshifting efficiency19 versus unfolding kinetics at the stretching forces of 0, 25, and 40 pN, respectively. The correlation is best at a stretching force of around 25 pN (see Supplementary Fig. S10). (f) In vitro frameshifting efficiency19 has no correlation with mechanical unfolding transition state position X≠. Error bars of both force and frameshifting efficiency represent standard deviations. Errors of lnk and X≠ are reported as standard errors.