Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways

Abstract

The ribosome is a massive ribonucleoprotein complex ( approximately 2.4 MDa) that utilizes large-scale structural fluctuations to produce unidirectional protein synthesis. Accommodation is a key conformational change during transfer RNA (tRNA) selection that allows movement of tRNA into the ribosome. Here, we address the structure-function relationship that governs accommodation using all-atom molecular simulations and single-molecule fluorescence resonance energy transfer (smFRET). Simulations that employ an all-atom, structure-based (Gō-like) model illuminate the interplay between configurational entropy and effective enthalpy during the accommodation process. This delicate balance leads to spontaneous reversible accommodation attempts, which are corroborated by smFRET measurements. The dynamics about the endpoints of accommodation (the A/T and A/A conformations) obtained from structure-based simulations are validated by multiple 100-200 ns explicit-solvent simulations (3.2 million atoms for a cumulative 1.4 micros), and previous crystallographic analysis. We find that the configurational entropy of the 3'-CCA end of aminoacyl-tRNA resists accommodation, leading to a multistep accommodation process that encompasses a distribution of parallel pathways. The calculated mechanism is robust across simulation methods and protocols, suggesting that the structure of the accommodation corridor imposes stringent limitations on the accessible pathways. The identified mechanism and observed parallel pathways establish an atomistic framework for interpreting a large body of biochemical data and demonstrate that conformational changes during translation occur through a stochastic trial-and-error process, rather than in concerted lock-step motions.

FIGURE 1.

Reversible fluctuations observed in simulations and smFRET. (A) Structure of the 70S ribosome with 16S rRNA (blue), 30S proteins (cyan), 23S rRNA (purple), 50S proteins (pink), mRNA (green), aa-tRNA (yellow), and p-tRNA (red) shown. aa-tRNA shown in the (B) A/T conformation and (C) A/A conformation. The purple dashed line, R_Elbow, is the distance between U47 of the aa-tRNA and U8 of the P-site tRNA (purple spheres), used experimentally, and computationally (distance between O3′ atoms), to measure aa-tRNA elbow accommodation. The orange dashed line, R_Arm, is the distance between the C3′ atom of G4 in the aa-tRNA and C5′ atom of G67 in P-site tRNA (orange spheres), which measures acceptor arm accommodation. The blue dashed line, R_3′, is the distance between A-site and P-site amino acids (blue spheres), which measures 3′-CCA end accommodation into the peptidyltransferase center. (D) Time trace of R_Elbow for a single accommodation simulation. (E) P(R_Elbow, t), the unnormalized probability, was calculated from 312 independent accommodation transitions (12 million sampled structures). Values are colored off-white (low) to red (high), on a log scale, throughout all figures. Since FRET efficiencies are inversely related to R_Elbow , the Y-axes in D and E are inverted for easier comparison with F and G. (F) Single-molecule time trace monitored after stop-flow delivery of cognate EF-Tu·GTP·Phe-tRNA^Phe(Cy5-acp³U47) to surface-immobilized ribosome complexes carrying fMet-tRNA^OH in the P-site (Cy3-s⁴U8). (G) FRET population histogram of single FRET traces postsynchronized to a FRET value of 0.323. Population only includes traces where the tRNA reaches the A/A state (N = 431). Note: Reversible fluctuations were observed in many simulations and FRET traces and each trace has a unique profile. The uncanny similarities between the shapes of D and F are coincidental.

FIGURE 2.

Probability distributions reveal sequential elbow, arm, 3′-CCA end accommodation. (A) P(R_Elbow, R_3′) shows four highly populated conformations: (1) A/T, (2) elbow accommodated, (3) acceptor arm accommodated, and (4) A/A states. Peaks are numbered consistently between plots, i.e., the same structures compose peak X (= 1,2,3,4) in each figure. (B) P(R_Elbow, R_Arm) prior to 3′-CCA entry into the PTC (R_3′ > 30 Å, above the black dotted line in A). The bend in the probability distribution, centered about peak 2, indicates sequential elbow-arm accommodation. Simultaneous elbow and arm accommodation events would fall on the gray dashed line, which connects peaks 1 and 3. (C) P(R_Arm, R_3′) for elbow-accommodated structures (R_Elbow < 30 Å, right of white dotted line in A). R_Arm and R_3′ are correlated, but the arm reaches the A/A conformation, while the 3′-CCA end is ∼40 Å from the PTC (peak 3). (D) Representative snapshot of the transition state associated with elbow accommodation (between peaks 1 and 2 in A), where the tRNA closely approaches H89. (E) Snapshot of the elbow-associated state (peak 2) colored as in Figure 1 with the A-site finger (gray) and A loop (pink) also shown and rotated 90° from D. (F) Representative structure of arm-accommodated state (peak 3).

FIGURE 3.

Displacement of H89 during aa-tRNA elbow accommodation. (A) Structure of A/T conformation with aa-tRNA (yellow), p-tRNA (red), mRNA (green), H89 (purple), and stem–loop of H89 (violet) shown. The distance between the stem–loop of H89 and the A-site codon (H89-codon), is indicated by the pink bar. The green bar indicates R_Elbow (as defined in Fig. 1B). (B) Probability distribution of H89-codon versus R_Elbow, calculated from 312 unrestrained structure-based simulations. Only structures in which the arm is not accommodated (R_Arm > 12 Å) are included in the distribution. The gray line is the average H89-codon distance as a function of R_Elbow. Note: X-axis is inverted for visual ease. As the aa-tRNA elbow accommodates (i.e., R_Elbow decreases) H89-codon increases when the aa-tRNA contacts the stem–loop of H89 (R_Elbow ∼ 49 Å) and then reduces after the aa-tRNA elbow has accommodated.

FIGURE 4.

Entropy from 3′-CCA flexibility resists accommodation. (A) 3′-CCA rotation coordinate, Φ₂ (Sanbonmatsu et al. 2005), formed by the O3′ atoms of residues 69, 71, 73, and O atom of Phe. (B) Probability as a function of Φ₂ and R_3′ indicates that in the A/T conformation (R_3′ ∼ 80–90 Å) the flexible 3′-CCA end samples all angles with relatively high probabilities. During acceptor arm accommodation (R_3′ = 40–60 Å), the restricted mobility of the 3′-CCA end indicates an entropic barrier. As the 3′-CCA end enters the PTC, the configurational entropy decreases as the 3′-CCA end proceeds via a (+) or (−) pathway (pink and black lines). Note that since Φ₂ is a dihedral angle, (B) is periodic along the Y-axis with a modulus of 360°. Representative snapshots of 3′-CCA entry (C) along the major groove of H89, (D) by passing over the A loop, or (E) by passing between H89 and the A loop.

FIGURE 5.

Comparison of structure-based simulations and explicit-solvent (ES) simulations. (A) Color contour plot is the probability distribution obtained from structure-based simulations. Transparent gray shadows delimit the region of the phase space sampled in 1.4 μs of explicit-solvent simulations (a slight shift of the A/A ensemble, to lower R_3′, is due to attraction between the aa-tRNA and p-tRNA amino acids). (B) Probability distribution from 704 TMD simulations with a structure-based force field. Two trajectories from explicit-solvent TMD (Sanbonmatsu et al. 2005) are shown as black and brown lines.