Structural characterization of mRNA-tRNA translocation intermediates

Abstract

Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.

Fig. 1.

Movement of domains and tRNAs. (A) Left to right, different superimposed cryo-EM densities of the 30S subunits from class 2 (yellow), class 4A (violet), class 4B (olive), and class 6 (gray). (B) Left to right, different reconstructions (upper row) and superimposed densities (lower row) of the 30S subunits; color-coded as in (A). The new orientation of the subunit is shown as a thumbnail on the left. The A- and P-site tRNAs are in magenta and green. (C) Left to right, close-ups of different reconstructions and superimposed cryo-EM densities of the 50S subunits; color-coded as in (A) and (B). The orientation of the subunits is shown as a thumbnail on the left. The arrows in (A), (B), and (C) indicate the directions of intersubunit rotation, head swivel, and L1 stalk movement, respectively. Colored labels identify the different class reconstructions.

Fig. 2.

Atomic models of tRNA obtained by flexible fitting. A gallery of images is shown for tRNA-mRNA complexes for classes as indicated in (A)–(F). Thumbnail in (A) shows the orientation of the tRNAs with respect to the 30S subunit (yellow). Cryo-EM densities for A- and P-site tRNAs are in transparent magenta and green. Fitted structures are in ribbons. Modeled mRNA sequence is shown in blue. (G) Stereo-views of fitted A and P-site tRNAs from class 2 (magenta and green), class 4B (olive), and class 6 (gray), aligned with respect to the 70S ribosomes (see colored labels). See also Fig. S4.

Fig. 3.

Tensor analysis of individual domains. (A) Trends of measured angles on the small subunit for this study (connected points), those obtained by Fu et al. (32) (NR, IRS1, IRS2, RS) and those by X-ray crystallography (PDB codes indicated). The values plotted, b/pl, H_tilt, H_oc, and H_sw, are as defined in Tables S2 and S3. (B) Progression of the L1 stalk closing. (L1_α and L1_β correspond to the same angle in an oblique plane). (C, D, E) Progression of tRNA and L1 movements. (F) Free-energy landscape along the intersubunit rotation coordinate, computed from occupancies of states in equilibrium. The numbers describe the relative stabilities of the various ground states. Dotted line represents the largely unknown topology of the energy landscape between the sample points obtained by cryo-EM. It includes a peak at 20 kcal/mol, going off scale in our diagram, indicating the barrier separating MS I and MS II as calculated from tRNA-tRNA smFRET measurements.