Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution

Abstract

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Fig. 1.

Cryo-EM reconstruction of eukaryotic 80S ribosomes. (A) T. aestivum and (B) S. cerevisiae 80S ribosomes, with small (40S) and large (60S) subunits colored yellow and gray, respectively and the P-tRNA, green. (C–F) Selected views of the T. aestivum 80S density map (blue mesh) and corresponding molecular model, with r protein in yellow and rRNA in white (backbone) and red (bases).

Fig. 2.

An atomic model for the T. aestivum 80S ribosome. (A and B) Secondary structures for the (A) small (18S) and (B) large subunit (5S, 5.8S, and 28S) ribosomal RNAs, with the newly modeled regions colored green. Expansion segments and variable regions are indicated in gray and unmodeled regions are orange. (C and D) Newly modeled regions of rRNA (green) are highlighted on the (C) small and (D) large subunit density map (Left) and as molecular models (Right). (E and F) Newly modeled proteins are highlighted on the (E) small and (D) large subunit density map (Left) and as molecular models (Right). Newly identified proteins are colored red, whereas de novo modeled extensions are colored light green, and modeled but unassigned proteins are yellow.

Fig. 3.

Ribosomal RNA expansion segments and variable regions. (A and B) Secondary structures for the T. aestivum (A) small (18S) and (B) large subunit (5S, 5.8S, and 28S) ribosomal RNAs, with the ES and variable regions (VR) colored distinctly. (C and D) Cryo-EM maps of the (C) small and (D) large subunits with assigned ES and VR colored as in A and B. (E and F) Molecular models of the ES and VR of rRNA colored as in C and D.

Fig. 4.

Molecular models for expansion segments ES3^S/ES6^S, ES7^L, and ES27^L. (A) Isolated density for ES6^Sd (blue) and ES3^Sa,c (gold) on the 40S subunit (Left) and transparent with a molecular model (Center). rRNA secondary structure prediction highlighting interaction between the loop of ES6^Sd and the bulge in ES3^Sc (Right), as proposed by ref. . (B) Isolated density for ES7^L from T. aestivum (T. a., blue) and S. cerevisiae (S. c., gold) on the 80S ribosome (Left) and transparent with a molecular model (Center). Ribosomal proteins L28e (red) stabilizes ES7^La in the T. aestivum 80S ribosome, whereas the extension of r-protein L6e appears to pass through the three-way junction formed by helices ES7^Lc–e (Right). Molecular models for the (gold) and (blue) positions (Left), as observed in S. cerevisiae 80S ribosomes (Thumbnail Insets) (39) and an intermediate position (, gray) observed in the T. aestivum 80S ribosome. In yeast, r-protein L34e (green) and L38e (red) interact with the and positions, respectively. The tunnel exit (TE) and L1 stalk (L1) are indicated for reference. (C) Schematic (Top Right) and molecular model (Middle Right) indicating that the interchange between the (gold) and (blue) positions involves a rotation of ∼110° of ES27^La–c relative to H63. Secondary structure for the junctions of S. cerevisiae ES27^La–c and H63.

Fig. 5.

Cryo-EM reconstructions of ribosomes from (A) the eubacterium Escherichia coli (31), (B) the yeast S. cerevisiae (27), (C) wheat germ T. aestivum (this work), and (D) Homo sapiens (44). The small and large subunits are shown in yellow and gray, respectively and the P-tRNA (green) is indicated for reference. The dashed lines and numbers indicate the number of nucleotides of the rRNA expansion segments that are not visualized.