Research ArticleBIOPHYSICS

Directional mechanical stability of Bacteriophage φ29 motor’s 3WJ-pRNA: Extraordinary robustness along portal axis

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Science Advances  26 May 2017:
Vol. 3, no. 5, e1601684
DOI: 10.1126/sciadv.1601684
  • Fig. 1 Schematic illustration of the 3WJ-pRNA and related SMD simulations.

    (A) The φ29 motor’s full-length pRNA, containing a core 3WJ structure and exterior motifs 1, 2, and 3. (B) The scheme used for loading force onto the helix termini. RNA strands A, B, and C are colored in red, green, and blue, respectively, in this and subsequent figures.

  • Fig. 2 Mechanical unfolding of 3WJ-pRNA under coaxial pulling conditions.

    (A) Force profile of the 3WJ-pRNA with and without bound Mg2+ ions. (B) The RMSD of the 3WJ-pRNA structures with (black) and without (red) bound Mg2+ ions under applied pulling forces. (C and D) Representative snapshots of the 3WJ-pRNA at t = 41.4 and 42.0 ns (that is, before and after the initial unfolding event near t = 41.5 ns).

  • Fig. 3 Rupture of Mg clamps during mechanical unfolding.

    (A) Coordination interactions of Mg2+ ions during unfolding. (B) Interphosphate separation between nucleotides G23 and A90 (black) and C24 and A89 (red) over the course of pulling. Inset: Zoom-in of the interphosphate separation near the rupture of the two Mg clamps.

  • Fig. 4 Mechanical unfolding of the 3WJ-pRNA under biomimetic conditions.

    (A) Schematic for the loading force onto the H1 terminus of the 3WJ-pRNA. Restrained nucleotides are shown in a van der Waals representation. (B) Representative configuration of the 3WJ-pRNA after initial unfolding. (C) Force profile of the 3WJ-pRNA with (black) and without (red) bound Mg2+ ions. Inset: Interphosphate distances associated with the two Mg clamps. (D) Survival curves for the unfolding of the 3WJ-pRNA under loading forces of 1600 and 2000 pN.

  • Fig. 5 AFM experiments of 3WJ-pRNA’s unfolding along H1-H3.

    (A) Schematic for AFM-based single-molecule force spectroscopy experiments. Both the cantilever tip and the glass substrate were modified with amine groups using APTES. Bifunctional PEG linkers were used to covalently link the 5′ amine–modified H1 to the substrate and H3 to the cantilever tip. (B) Two typical force-extension curves for the rupture of the 3WJ-pRNA in TMS buffer [89 mM tris, 5mM MgCl2 (pH 7.6)]. (C) Rupture force histogram for traces shown in (B). (D) Rupture force histogram for the unfolding in tris buffer [89 mM tris, 20 mM EDTA (pH 7.6)]. (E) Dependence of the rupture force on the loading rate for the unfolding in TMS buffer.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1601684/DC1

    discussion S1. Divalent ion models studied in this work and the corresponding results.

    discussion S2. Extrapolation of lifetime of folded state to the loaded force of 20 pN from SMD simulations and AFM experiments

    table S1. The dimension of simulation systems.

    table S2. The parameters of divalent ions.

    fig. S1. Force profiles during the unfolding along H1-H3 (16 trajectories).

    fig. S2. Structural analysis of 3WJ-pRNA during the unfolding along H1-H3.

    fig. S3. Force profiles of 3WJ-pRNA during the unfolding along H1-H3 (eight trajectories).

    fig. S4. Force profiles during the unfolding along H1-H3 (pulling rate is 1 nm/ns).

    fig. S5. Cooperativity of two Mg clamps during mechanical unfolding.

    fig. S6. Force profiles during the unfolding along H1-H3 using the Merz model of Mg2+ ions.

    fig. S7. Relaxation and mechanical unfolding of 3WJ-pRNA using the Aqvist model of Ca2+ ions.

    fig. S8. Relaxation and mechanical unfolding of 3WJ-pRNA using the Merz model of Ca2+ ions.

    fig. S9. Mechanical unfolding of 3WJ-pRNA under transverse (H1-H2) pulling.

    fig. S10. Mechanical unfolding of 3WJ-pRNA under transverse (H2-H3) pulling.

    fig. S11. Force profiles of 3WJ-pRNA with force applied to the termini of H1 at a pulling rate of 0.1 nm/ns.

    fig S12. Representative length-time traces of 3WJ-pRNA when constant force is loaded onto the terminus of H1.

    fig S13. Distribution of unfolding times for the pRNA-3WJ under the loading forces of 1600 and 2000 pN.

    fig. S14. Dependence of the rupture force on the loading rate.

    fig. S15. Schematic of 3WJ-pRNA in the crystal structure (4KZ2), where three base pairs (bold) are attached to the terminus of H3.

    References (5456)

  • Supplementary Materials

    This PDF file includes:

    • discussion S1. Divalent ion models studied in this work and the corresponding results.
    • discussion S2. Extrapolation of lifetime of folded state to the loaded force of 20 pN from SMD simulations and AFM experiments
    • table S1. The dimension of simulation systems.
    • table S2. The parameters of divalent ions.
    • fig. S1. Force profiles during the unfolding along H1-H3 (16 trajectories).
    • fig. S2. Structural analysis of 3WJ-pRNA during the unfolding along H1-H3.
    • fig. S3. Force profiles of 3WJ-pRNA during the unfolding along H1-H3 (eight trajectories).
    • fig. S4. Force profiles during the unfolding along H1-H3 (pulling rate is 1 nm/ns).
    • fig. S5. Cooperativity of two Mg clamps during mechanical unfolding.
    • fig. S6. Force profiles during the unfolding along H1-H3 using the Merz model of Mg2+ ions.
    • fig. S7. Relaxation and mechanical unfolding of 3WJ-pRNA using the Aqvist model of Ca2+ ions.
    • fig. S8. Relaxation and mechanical unfolding of 3WJ-pRNA using the Merz model of Ca2+ ions.
    • fig. S9. Mechanical unfolding of 3WJ-pRNA under transverse (H1-H2) pulling.
    • fig. S10. Mechanical unfolding of 3WJ-pRNA under transverse (H2-H3) pulling.
    • fig. S11. Force profiles of 3WJ-pRNA with force applied to the termini of H1 at a pulling rate of 0.1 nm/ns.
    • fig S12. Representative length-time traces of 3WJ-pRNA when constant force is loaded onto the terminus of H1.
    • fig S13. Distribution of unfolding times for the pRNA-3WJ under the loading forces of 1600 and 2000 pN.
    • fig. S14. Dependence of the rupture force on the loading rate.
    • fig. S15. Schematic of 3WJ-pRNA in the crystal structure (4KZ2), where three base pairs (bold) are attached to the terminus of H3.
    • References (54–56)

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