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Overview of P/E hybrid formation. Before hybrid-state formation (a)–(d), the ribosome (grey and cyan) adopts an unrotated configuration, where the tRNAs are in classical P/P (red) and A/A (yellow) configurations (mRNA in green). In the P/E hybrid configuration (e)–(h), tRNA molecules are in the P/E and A/P* 76 configurations (f), and the small subunit of the ribosome is rotated by ∼7° (h). To measure movement of tRNA through the ribosome, we calculated the distance between the tRNA elbow (R Elbow , defined previously 33,53 ), which changes from 36 Å to 58 Å during the transition. Movement of the 3′-CCA end of the P-site tRNA is measured by the center of mass distance between the side chain of A76 and A2411/A2412 of the 23S rRNA: R CCA−E , which is 47 Å and 2 Å in the P/P and P/E configurations. Rotation of the 30S body is measured by a collective reaction coordinate θ body (defined previously 54 ), which is 0° and 7° in the classical and P/E hybrid configurations. For visual clarity, proteins are not shown, though all simulations also included the ribosomal proteins.
Schematic of the potential energy surface used to simulate hybrid-state formation. We constructed a multi-basin model, where each tRNA can be stably associated with multiple ribosomal binding sites. During P/E formation, the relevant interactions are with the P and E sites. When forming a hybrid configuration, the tRNA and ribosome are in an energetically strained configuration, where the P/P configuration is of lower energy than the P/E. Q denotes a structural reaction coordinate that can capture the endpoints and transition-state ensemble.
Movement along multiple coordinates is not simultaneous. During simulations of hybrid-state formation, large changes occur along the (a) elbow, (b) 3′-CCA and (c) 30S body rotation coordinates. Since the simulations employed a restraint-based form of TMD with a small energetic weight, spontaneous fluctuations are observed in each simulation (e.g., fluctuations of >10 Å are observed in the 3′-CCA end. See panel (b) and inset). Probability distributions along multiple coordinates highlight sequential rearrangements and suggest barriers. (d) Initially, the 30S body rotates, with minimal movement of the tRNA, which is followed by rapid movement of the elbow. (e) The tRNA elbow moves nearly 20 Å towards the E site before the 3′-CCA end releases from the P site. During this transition, there is a subtle peak that forms at R CCA − E ∼ 20 Å. The presence of an intermediate ensemble (IE) is indicative of a steric (H74), or entropic barrier during hybrid formation. (f) The width of the distribution of 3′-CCA angles (measured by ∅2 33,46 ) is roughly uniform as the 3′-CCA end moves towards the E site. This is in contrast to tRNA accommodation, where changes in the distribution of angles is significantly larger. 77
Structural characteristics of tRNA elbow movement during hybrid formation. (a) Initially, the tRNAs are in classical configurations, where the 30S subunit is in an unrotated orientation. (b) The majority of the subunit can rotate, with minimal movement (several Å) of the tRNA elbow. (c) Structural representation of residues that the tRNA elbow interacts with during hybrid formation. (d) Same as (c), with the P-site tRNA not shown. Residues that the tRNA contacts in more than 10% of the simulations are shown in stick representation. The coloring of the contacted residues ranges from red (contacted in 10% of the runs) to blue (contacted in 100% of the runs). As the tRNA elbow moves between the P and E sites of the large subunit, few interactions are formed between the elbow and ribosome. With the exception of interactions near the P site and with the L1 stalk, the tRNA only contacts protein L33. The minimal number of contacts is consistent with the absence of a pronounced peak in the probability distribution along R Elbow and θ body (Figure 3(d) ).
Structural characteristics of tRNA 3′-CCA end movement during hybrid-state formation. (a) As the hybrid configuration is adopted, the tRNA elbow reaches a near-P/E position before the 3′-CCA end dissociates from the P site. (b) P/E configuration, shown for comparison. Panels (c)–(e) depict which residues are contacted by the 3′-CCA end during hybrid formation. The representation is consistent with Figure 4 . (c) All residues that are contacted are shown, as well as the P/P-configured tRNA. (d) Only the rRNA residues that are contacted are depicted. The 3′-CCA end is shown for reference. (e) Protein residues that are contacted are shown. The most dominant region of interaction is H74, which may serve as a guide, upon which the 3′-CCA end moves. In many simulations, the 3′-CCA end enters a pocket formed by residues near A2426 and H74, suggesting that H74 also gives rise to a significant steric barrier.
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