Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome

Abstract

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Figure 1. Statistical distribution of particle resemblance

Histogram of values obtained by forming the difference between correlation coefficients of each experimental projection with respect to two references. A reference line is drawn at the selected cutoff point (ΔCC=0). The data were sorted based on their resemblance to 3D template ribosome maps that differ in the ratcheting rotation of the 30S subunit (insets). Both reference volumes were from a previous study (Valle et al., 2003b). Reference 1 was the cryo-EM density map of a pre-translocational complex (70S-tRNA^fMet-fMet-Phe-tRNA^Phe), while reference 2 was the cryoEM map of a ribosomal complex bound with EF-G-GDPNP (70S-MFTI-tRNA^Ile-EF-G-GDPNP + puromycin), which displayed ratchet like rotation. The densities corresponding to tRNAs and EF-G were computationally removed to avoid reference bias. To this end, soft Gaussian masks were used. Using this strategy, a clear bimodal distribution was obtained and two major ribosomal populations were separated. One, composed by 57,603 particles, corresponded to unratcheted ribosomes bearing the classic A/A-P/P tRNA configuration, while the second population, consisting of 158,969 particles, showed the ratched-like rotation of the 30S subunit hand-in-hand with the A/P-P/E hybrid tRNA configuration as shown in Figure 2.

Figure 2. General features of classic and hybrid ribosomes

Cryo-EM densities are shown for the small (yellow) and large (blue) subunits, and A and P-site tRNAs (magenta and green, respectively). The classic-state ribosome is shown in (A) and the hybrid-state ribosome in (B). Labels and landmarks: sp (spur), bk (beak), h (head), CP (central protuberance), L1 (L1 stalk), L7/L12 (L7/L12 stalk).

Figure 3. Conformational changes upon ratcheting movement and tRNA reconfiguration

(A) Superimposition of aligned 30S (left) and 50S (right) subunits shown from the intersubunit side. Classic-state subunits are shown in transparent grey, whereas the subunits of the hybrid-state ribosome are shown in solid yellow (30S) and blue (50S). (B) Close-up view of the 50S subunit showing the classic tRNA configuration. (C) Close-up view of the 50S subunit showing the hybrid tRNA configuration. The orientations of the subunits are shown as successive thumbnails on the left. Labels and landmarks: S19 (small subunit/SSU protein S19), S13 (SSU protein S13), h23 (SSU helix 23), h44 (SSU helix 44), H38 (large subunit/LSU helix 38), H68 (LSU helix 68), H69 (LSU helix 69), H76 (LSU helix 76), L5 (LSU protein L5). All other landmarks have been introduced in Figure 1.

Figure 4. Hybrid configuration of the ribosome and implications for EF-G based translocation

(A) Superimposition of aligned 30S subunits from hybrid (solid yellow), classic (transparent grey) and EF-G bound (transparent purple) forms (from Valle et al., 2003b). In the right panel, the binding position of EF-G (red) is shown. (B) Close-up view of the superimposition of hybrid (solid yellow) and EF-G bound (transparent purple) forms in the presence of EF-G and hybrid tRNAs. The subunits are shown from the intersubunit side.

Figure 5. Comparison between the positions and orientations of classic and hybrid tRNAs deduced from the cryoEM maps

The fitting of the experimental densities with the X-ray structures of the tRNAs by real space refinement using rigid body fitting. Although the CCA-end of the classic A/A tRNA was not well-defined in the experimental density map, the complete quasi-atomic structure is shown for clearer visualization and perception of the relative positions of the different configuration tRNAs. The structures are displayed in ribbons.