Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates

Abstract

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.

Figure 1

Molecular architecture of late pre-40S ribosomes. (A) SDS-PAGE analysis of pre-40S particles purified via Rio2-TAP. (B) 18Å cryo-EM reconstruction of pre-40S particles purified via Rio2-TAP. (C) Densities of AFs (in orange) revealed after explicit-solvent MDFF of the structure of mature 40S ribosomes into the cryo-EM map. rRNA is shown in white, Rps are shown in graphite and are annotated as in (13). H44 and H45 are highlighted in magenta. Subunit (left) and solvent (right) interface views are shown. (D) H44 is distorted in pre-40S particles. Rigid-body docking of the mature H44 structure (cyan) does not fit the corresponding density in the cryo-EM map (gray). MDFF allows for improved fit of H44 (magenta), accompanied by a change in the positioning of the decoding site residues (yellow). Close-up view reveals that the distance between G577 and A1755 is increased from 3 to ~4.5 nm in the pre-40S (magenta) or mature 40S (cyan) ribosomes.

Figure 2

(A) SDS-PAGE analysis of wild-type Rio2TAP, Gal1∷Nob1, Δ-Ltv1, wild-type Ltv1TAP, Gal1∷Rio2 and Gal1∷Tsr1 used in the cryo-EM shows depleted and co-depleted proteins. (B) cryo-EM maps for the wild type, Gal1∷Nob1, Δ-Ltv1 (solvent view) and wild type, Gal1∷Rio2, Gal1∷Tsr1 (subunit view) pre-40S particles identify densities belonging to individual assembly factors (See also Fig. S5–S14). Colored arrows point to the missing densities with color-coding as in (C). (C) Positioning of AFs on pre-40S particles. The structures of archeal Rio2 (blue) and human Dim1 (green) are docked within the corresponding cryo-EM densities.

Figure 3

AFs obstruct translation initiation factor binding sites, and prevent mRNA binding. (A) Tsr1, Rio2 and Dim1 block binding of eIF1 and eIF1A. Sites of RNA footprints from Fe-labeled eIF1 and eIFA are shown in red and blue, respectively (44, 45). (B) Nob1 and Pno1 block binding of eIF3. Nob1 and Pno1 densities are shown in orange and red, respectively. The density for eIF3 (46) is shown in purple. (C) The latch between H18 and H34 closes the mRNA channel in mature (26) (blue) and pre-40S ribosomes (grey). The Tsr1 density was removed here to allow for better visualization of the latch. (D) Overlay of the structure of mature 40S ribosomes with eIF1 and eIF1A bound (26) (in aqua) and pre-40S particles (grey) shows that the hinge on the back of the beak (aqua) and the density for the Enp1/Ltv1/Rps3 complex (yellow) partially overlap. RACK1 is present only in mature 40S and its density is indicated with an asterisk.

Figure 4

Tsr1 blocks 60S subunit joining. (A) Peptides from ribosomal proteins belonging to the large subunit (Rpl; identified by mass spectrometry) copurify heavily with pre-40S particles purified from Gal1∷Tsr1 strains, but not from the wild type control, or the Gal1∷Rio2 strain. (B) 3D cryo-EM reconstructions confirm that larger particles in Ltv1TAP purifications from Gal1∷Tsr1 cells are 80S particles. (C) Northern blotting shows that 25S rRNA from the large subunit copurifies with pre-40S particles in Gal1∷Tsr1 cells relative to wild type or Gal1∷Rio2 cells. (D) Western blot of fractions from 10–50% sucrose gradients demonstrates that Ltv1TAP shifts from being bound only to 40S-preribosomes to 80S fractions when Tsr1 is depleted, but not when Rio2 is depleted. (E) Northern probing on gradient fractions demonstrates that 80S fractions in the Tsr1 depleted strain contain 20S and 25S rRNA, but not 35S or 23S pre-rRNA. The position of 40S, 60S and 80S fractions determined by absorbance is indicated. Fig. S19 shows the absorbance profiles from these gradients.