Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Abstract

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Fig. 1.

Identification of r-proteins L38e and L34e. (A) Cryo-EM map of the T. aestivum 80S ribosome, with rRNA colored gray and r protein colored green. (B) Same as A, but with localized r proteins colored red. Reconstruction of (C) S. cerevisiae WT 80S ribosome compared to (D) reconstruction of S. cerevisiae 80S ribosomes isolated from a strain lacking the gene for L38e. The asterisk indicates the position of additional density assigned to L38e, and the tunnel exit (TE) is shown for reference. (E) Difference density map calculated between C and D and shown superimposed on the map from D. Reconstruction of (F) P. furiosus 70S ribosome, compared to (G) X-ray structure of the 50S subunit from H. marismortui filtered to a similar resolution. (H) Difference density map calculated between F and G and shown superimposed on the map from G identifying the location of r-protein L34e (red).

Fig. 2.

Localization of eukaryote-specific r proteins. Cryo-EM maps of the T. aestivum (A) 40S and (B) 60S subunit, with density for the newly identified r proteins colored distinctly. Molecular models of r proteins of the T. aestivum (C) 40S and (D) 60S subunit, with newly identified r proteins colored distinctly.

Fig. 3.

Functional roles for eukaryote-specific r proteins. (A) Small 40S subunit with newly modeled r-proteins S30e and S25e (red) and eukaryote-specific extension of S4p (green) highlighted (thumbnail, Left; zoom, Right). (B) Comparative view of the bacterial 30S subunit decoding site (11, 12). In A and B, the anticodon-stemloops of A-, P- and E-tRNAs (blue) and mRNA (orange) are shown for reference. (C) Large 60S subunit with eukaryote-specific extension of L10e (green) highlighted (thumbnail, Left; zoom, Right). (D) Comparative view of the bacterial 50S subunit with bacterial-specific L27p colored red (11). In C and D, the acceptor-stem of the P-tRNA (blue) is shown for reference. (E) Small 40S subunit with newly modeled r-proteins S21e, S26e, and S28e colored distinctly (thumbnail, Left; zoom, Right). (F) Comparative view of the bacterial 30S subunit with bacterial-specific S18p shown in green (11). In E and F, the P-tRNA (blue) and mRNA (orange) are shown for reference. (G and H) The binding site of eEF3 on the S. cerevisiae 80S ribosome, with (G) side and (H) top views (see insets) showing the binding site of eEF3 as a red outline and molecular models of ribosomal components that comprise the eEF3 binding site. Newly identified proteins are shown in red (S19e, S25e) and newly modeled r-protein extensions in green, whereas core r proteins are colored gray. Modified from ref. .

Fig. 4.

Coevolution of rRNA expansion segments with r proteins in the 80S ribosome. (A) Cryo-EM map of the T. aestivum 80S ribosome, with rRNA ES and variable regions colored green and eukaryote-specific r proteins and extensions colored orange. (B) View of the intertwined region of ES7^L (dark blue) and ES39^L (light blue), with core r proteins (gray), eukaryote-specific r-protein extensions (pale green), and r proteins (L6e, orange; L14e, red; L18ae, yellow; L28e, pink; L35ae, green) highlighted. Inset shows relative position to 40S (yellow) and 60S subunits (gray). (C) Comparison of relative positions of S4e (red) in yeast/T. aestivum 80S (Left) with S16p (green) in bacteria (11) (Right). (D) Comparison of relative positions of L29e (red) in yeast/T. aestivum 80S (Left) with L36p (green) in bacteria (11) (Right).

Fig. 5.

Structures of wheat germ and yeast eukaryotic 80S ribosomes. (A and B) Near-complete molecular models for the (A) T. aestivum and (B) S. cerevisiae 80S ribosome, with rRNA and protein shown in yellow and orange for the small subunit and gray and blue for the large subunit, respectively.