Ribosome Assembly Factors Prevent Premature Translation Initiation by 40S Assembly Intermediates

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Science  09 Sep 2011:
Vol. 333, Issue 6048, pp. 1449-1453
DOI: 10.1126/science.1208245


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.

In eukaryotes, the assembly of the ribosomal subunits from the four ribosomal RNAs (rRNAs) (18S, 5.8S, 25S, and 5S) and 78 ribosomal proteins is facilitated by a conserved macromolecular machinery comprising ~200 assembly factors (AFs). These proteins, which are mostly essential, catalyze the modification, cleavage from precursor transcripts, and folding of the rRNA and also facilitate the binding of ribosomal proteins (1). Although the components of this assembly line have been identified, their functions remain largely unknown. Pioneering electron cryo-microscopy (cryo-EM) studies (27) followed by crystal structures of prokaryotic (811) and eukaryotic mature ribosomes (12, 13) have provided functional insight into their complex architecture. However, little is known about the structure of assembly intermediates or the binding sites for AFs.

Whereas most ribosome assembly steps take place in the nucleolus, where rRNA is transcribed, final maturation of both subunits occurs in the cytoplasm. There, assembling ribosomal subunits encounter large pools of mature 40S and 60S subunits, mRNA, and translation factors, which presents a unique challenge: preventing premature translation initiation on immature subunits. This is of particular concern for assembling 40S subunits, as translation initiation factors, mRNAs, and the 60S subunit all bind to 40S subunits to initiate translation. Furthermore, premature 40S ribosomes, when incorporated into 80S ribosomes, are rapidly degraded (14). These considerations suggest the existence of mechanisms to prevent 80S formation before 40S maturation is complete.

We used cryo-EM to determine the structure of a late pre-40S ribosome assembly intermediate at 18 Å resolution, purified from Saccharomyces cerevisiae via TAP-tagged Rio2, an essential kinase required for 18S rRNA production (Fig. 1 and fig. S1). As expected (15), this particle includes six additional AFs: (i) the methylase Dim1; (ii) the endonuclease Nob1, which produces the mature 3′-end; (iii) its regulator Pno1 (also referred to as Dim2); (iv) the guanosine triphosphatase (GTPase)–like protein Tsr1; (v) the export adaptor Ltv1; and (vi) Enp1, a protein of unknown function (Fig. 2A). Furthermore, all small-subunit ribosomal proteins (Rps) except Rps10 and Rps26 are present (table S1). The three-dimensional (3D) cryo-EM reconstruction, as well as 2D averages of negative-stained particles (fig. S2), show all features of the mature 40S subunit: the head, beak, platform, and left and right foot (16). For a better interpretation of the EM densities, we applied explicit-solvent molecular dynamics flexible fitting (MDFF) (1720) to fit the structure of the mature 40S ribosome into the cryo-EM map (figs. S3 and S4), and we subsequently subtracted the resulting model from our reconstruction. This revealed the densities corresponding to AFs (orange in Fig. 1A), which are located on the subunit interface, at the back of the beak, and on the back of the platform. All of these regions are important for translation initiation. In addition, MDFF allowed us to characterize a major shift in conformation between mature and pre-40S particles at the upper part of helix 44 (H44), which affects the decoding site region (Fig. 1B).

Fig. 1

Molecular architecture of late pre-40S ribosomes. (A) Model of mature 40S fit by MDFF into the 18 Å cryo-EM reconstruction (transparent envelope) of pre-40S particles purified via Rio2-TAP. Orange, densities of AFs; white, rRNA; gray, Rpss [annotated as in (13)]; magenta, H44 and H45. (B) H44 is distorted in pre-40S particles. (Left) Rigid-body docking of the mature H44 structure (light blue) 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). (Right) 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 (blue) ribosomes.

Fig. 2

(A) SDS–polyacrylamide gel electrophoresis analysis of WT Rio2TAP, Gal1::Nob1, Δ-Ltv1, WT Ltv1TAP, Gal1::Rio2, and Gal1::Tsr1 used for cryo-EM shows depleted and codepleted proteins. (B) Cryo-EM maps for wild type, Gal1::Nob1, Δ-Ltv1 (solvent view) and wild type, Gal1::Rio2, Gal1::Tsr1 (subunit view) pre-40S particles reveal the densities belonging to individual assembly factors (see also figs. S5 to S14). Colored arrows point to the missing densities [color-coding as shown 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.

To assign the extra densities to individual AFs, we individually depleted (or deleted) the assembly factors Nob1, Rio2, Tsr1, and Ltv1 and determined the cryo-EM structures of the resulting particles to resolutions of 20, 22, 26, and 20 Å, respectively (Fig. 2, A and B, and figs. S5 to S10). Comparisons, including t-test difference mapping, of the wild-type (WT) cryo-EM map with maps of particles lacking individual AFs allowed for localization of factors. For example, in the Nob1 depletion, Nob1 was the only missing protein, allowing us to unambiguously assign its density at the platform. In contrast, in the Rio2 depletion, Nob1 and Dim1 were also missing. In that case, we used previous footprinting data to assign Dim1 and Rio2. The crystal structures for Dim1 and Rio2 fit well into their respective densities with cross-correlation values of 0.854 and 0.904, respectively (Fig. 2C and fig. S8). In addition, we obtained a 30 Å 3D reconstruction of negative-stained recombinant Tsr1 (figs. S11 and S12) and employed antibody labeling against Ltv1 and Enp1 (figs. S13 and S14) to substantiate our assignments for Tsr1, Ltv1, and Enp1 [see (21) for a detailed discussion on AF assignment]. These results are summarized in Fig. 2C. Importantly, these placements are largely consistent with previous cross-linking data, as well as a systematic analysis of protein-protein interactions (figs. S15 and S16) (2126).

The cryo-EM maps show that the AFs on the subunit interface (Rio2, Tsr1, and Dim1) overlap the binding sites of translation initiation factors eIF1 and eIF1A (Fig. 3A), as previously shown for the bacterial homologs of Dim1 and eIF1 (26). Furthermore, the binding sites for the nuclease Nob1 and its regulator Pno1 overlap the binding site for eIF3 at the platform (Fig. 3B). Thus, recruitment of initiation factors that directly bind to the 40S subunit is prevented by the joint activity of Nob1, Pno1, Tsr1, Dim1, and Rio2.

Fig. 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 (45, 46). (B) Nob1 and Pno1 block binding of eIF3. Nob1 and Pno1 densities are shown in orange and red, respectively. The density for eIF3 (47) is shown in purple. (C) The latch between H18 and H34 closes the mRNA channel in mature (27) (blue) and pre-40S ribosomes (gray). 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 (27) (in aqua) and pre-40S particles (gray) 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; its density is indicated with an asterisk.

During translation initiation, recruitment of the mRNA to the 40S subunit is facilitated by the activity of eIF1 and eIF1A (27). Their joint binding promotes a conformational switch in the 40S subunits that breaks the latch between helix 18 and helix 34 and opens the mRNA channel (Fig. 3C). mRNA channel opening is stabilized on the solvent side by a hinge formed between the beak and shoulder region (Fig. 3D) (27). The Enp1/Ltv1/Rps3 complex overlaps the hinge formed upon mRNA channel opening (Fig. 3D) and probably inhibits breaking of the latch in the pre-40S particles (Fig. 3C).

Despite the abundance of mRNAs and 60S subunits, 80S ribosomes are rarely (<10% of particles) observed in pre-40S purifications. Depletion of Tsr1 from the pre-40S particle increases the abundance of peptides of 60S ribosomal proteins that copurify with pre-40S subunits more than sevenfold, such that the number of 60S peptides is similar to that of 40S peptides (Figs. 2A and 4A). In addition, the amount of 25S rRNA is increased 20-fold (Fig. 4C), and 80S particles are frequently observed in cryo-EM images (Fig. 4B). To confirm that depletion of Tsr1 leads to formation of 80S ribosomes containing pre-40S–bound Ltv1 in vivo, we carried out sucrose gradient analysis to separate free proteins from 40S, 80S, and polysome fractions and probed for Ltv1 localization. As expected, depletion of Tsr1, but not Rio2, shifts Ltv1 from being exclusively bound to a 40S-like particle to also being bound to an 80S particle, indicative of 60S joining (Fig. 4D). Northern blot analysis confirms that this 80S particle contains 20S and 25S rRNAs, indicating that it is not a 90S pre-ribosome, which would contain 35S rRNA but not 25S or 20S rRNAs (Fig. 4E). Tsr1 and Pno1 are both depleted in the Tsr1 depletion but are present in all other purifications and therefore could both contribute to this effect. Alternatively, the loss of all interface-binding AFs together could also be responsible for 60S joining. Pno1, but not Tsr1, can be found in polysome fractions (28, 29), suggesting that the presence of Pno1 does not antagonize 80S formation. Depletion of human Tsr1 leads to increased turnover of cytoplasmic 40S pre-ribosomes, as expected when pre-40S ribosomes form inactive 80S ribosomes, which are degraded by the no-go pathway (30). Thus, Tsr1 (perhaps together with Dim1 and Rio2) inhibits the premature association of 60S subunits with assembling 40S subunits in the cytoplasm. This anti–subunit-association activity of Tsr1 is analogous to that performed by eIF6 and Nmd3 for late cytoplasmic 60S precursors (3133). However, Tsr1 does not provide a complete block to 60S joining, as low amounts of 60S subunits copurify with WT pre-40S subunits, consistent with the previous observation that low amounts of Nob1 and 20S rRNA can be found in polysomes (14).

Fig. 4

Tsr1 blocks 60S subunit joining. (A) Peptides from 60S ribosomal proteins (identified by mass spectrometry) copurify heavily with pre-40S particles purified from Gal1::Tsr1 strains, but not from the WT control or the Gal1::Rio2 strain. (B) Three-dimensional cryo-EM reconstructions confirm that larger complexes 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 WT or Gal1::Rio2 cells. (D) Western blot of 10 to 50% sucrose gradient fractions demonstrates that Ltv1TAP is also bound to 80S fractions when Tsr1 is depleted, but not when Rio2 is depleted. (E) Northern blot 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 positions of 40S, 60S, and 80S fractions determined by absorbance are indicated. Absorbance profiles from these gradients are shown in fig. S19.

During decoding, adenosines 1755 and 1756 (A1755/A1756) flip out of H44, and guanosine 577 (G577) rotates around the glycosidic bond to interrogate the mRNA/tRNA helix by hydrogen bonding with the minor groove (34). The cryo-EM map of the pre-40S particle shows that H44 is kinked, which drives the upper portion outwards and to the left (from the perspective of the 60S subunit). As a result, the nucleotides in the decoding site are displaced from their location in mature 40S subunits (Fig. 1B). A1755/A1756 and G577 move in opposite directions (Fig. 1B) so that the distance between them increases, precluding interactions of all three nucleotides with the mRNA/tRNA duplex. Hence, the shift in H44 disrupts the decoding site and prevents subsequent steps leading to peptide bond formation.

Thus, all seven late-binding cytoplasmic ribosome AFs contribute to a redundant and multipronged approach to chaperone pre-40S subunits and prevent them from prematurely engaging the translational apparatus. In this model, binding of translation factors eIF1, eIF1A, and eIF3 is precluded by binding of Dim1, Tsr1, Rio2, Pno1, and Nob1, whereas opening of the mRNA channel is prevented by binding of Enp1 and Ltv1 (Fig. 3). Tsr1 blocks the premature binding of 60S subunits (Fig. 4), and the decoding site is disrupted as a result of a prominent kink in H44 (Fig. 1B). Bacteria lack all of these factors, with the exception of Dim1, which blocks binding of IF3, the homolog of eIF1 (26). However, during 30S assembly in bacteria, the GTPase Era binds to the anti–Shine-Dalgarno sequence (35). Because bacterial mRNA recruitment occurs via base pairing between Shine-Dalgarno and anti–Shine Dalgarno sequences, binding of Era might prevent mRNA recruitment (35) analogous to Enp1 and Ltv1, although by entirely different mechanisms. Overlapping the function of Era is RbfA, whose binding to the bacterial 30S subunit leads to a deformation in H44 similar to the one observed here. Interestingly, the binding site for RbfA is similar to the Pno1 binding site (36). Thus, it seems possible that the function of AFs is conserved between kingdoms, but different proteins have evolved to maintain these functions, reflecting the differences in the translation initiation pathways. The lack of redundancy in prokaryotes might reflect the fact that 30S assembly intermediates in polysomes are not as efficiently degraded in bacteria (37) as they are in eukaryotes (14).

Rps10 and Rps26 are the only ribosomal proteins that are not yet bound to the late pre-40S particles examined here (table S1), consistent with the observation that their depletion does not stall processing of the 18S precursor, 20S rRNA (38). Binding of Rps26 to 20S rRNA is negligible and does not increase when 20S rRNA is accumulated, indicating that Rps26 binds after formation of 18S rRNA (39). Rps26 binds on the platform (13), where it overlaps the Pno1 binding site in the pre-40S particle (fig. S17A), indicating that Rps26 cannot bind before Pno1 dissociates. Similarly, Rps10 binds to the beak, overlapping the binding sites for Ltv1/Enp1 (fig. S17B). Incorporation of these proteins might be a final regulated step in 40S assembly.

Although Rps3 is present in pre-40S particles (table S1), the cryo-EM map shows that it does not occupy its final position (fig. S17C). This finding is consistent with previous data suggesting that in this pre-40S particle, but not in mature 40S ribosomes, Rps3 is salt-labile (15). Additionally, Rps14 is repositioned on the platform, consistent with previous findings that the interactions of its C-terminal extension with rRNA form late during ribosome assembly (40). The late assembly of both the platform region, where Rps26 and Rps14 bind, as well as the head/beak region, where Rps10 and Rps3 bind, is paralleled in the in vitro assembly of the bacterial 30S subunit, where the head and platform region are the last to form, and intermediates lack the homologs for Rps14, Rps26, and Rps3 (41).

Nob1, the nuclease for cleavage at the 3′-end of 18S rRNA, is present in the late pre-40S particle purified in our study; yet the particle only contains 20S rRNA (Fig. 4B). Thus, either an external signal is required to activate Nob1, or Nob1 is mispositioned in the presence of the other AFs and, therefore, inactive. In vivo and in vitro footprinting show that Nob1 protects the active site (42), and the EM map shows that Nob1 is close to the cleavage site (Fig. 2), suggesting that any rearrangement, if it occurs, is limited. The C-terminal extensions of Rps14 and Rps5 are important for D-site cleavage and engage rRNA immediately before that step (40, 43).

Previous work has shown that the bacterial homolog for Dim1 is inhibited by ribosomal protein S21. Although bacterial S21 has no eukaryotic sequence homolog, its binding site is almost identical to that of Rps26, suggesting that Rps26 is a functional homolog of S21 (13). Pno1 occupies the Rps26 binding site in pre-40S particles (fig. S17A). Because Pno1 binds directly to Nob1 and affects its interaction with rRNA (25), reverse communication from the Dim1 site could reposition Pno1, thus regulating Nob1. Dim1 is positioned to receive signals regarding assembly of the head/beak region, which is shown here to be incomplete, as Rps10 is absent and Rps3 is not fully incorporated. Binding of the bacterial homologs for Rps3, and its direct binding partners Rps20 and Rps29, to the head stabilizes an RNA conformation that inhibits the activity of Dim1’s bacterial homolog at the subunit interface (44). Hence, completion of head assembly could trigger changes at the Dim1 site, which could be relayed to reposition Pno1 and Nob1 for final pre-rRNA cleavage.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S24

Tables S1 to S5

References (4883)

References and Notes

  1. A detailed discussion of factor assignment and materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank M. Campbell for the gift of recombinant Tsr1 and (MBP)Tsr1; J. P. Gélugne for yeast strains; J. Doudna for making eIF3 maps available; H. Remmers (Univ. of Michigan) and M. Chalmers (The Scripps Research Institute–Florida) for performing mass spectrometry experiments; S. Ludtke, J. Zhang, and L. Passmore for EMAN scripts to generate variance maps; and J. Doudna, J. Cleveland, K. Nettles, T. Walz, and members of the Karbstein and Skiniotis labs for comments. B.S.S. is supported by a NSF predoctoral fellowship. This work is supported by NIH grant R01-GM086451 (to K.K.), the NIH-supported resource Multiscale Modeling Tools for Structural Biology (grant RR12255 to C.L.B.), and the Univ. of Michigan Biological Sciences Scholars Program (G.S.). EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-1922, -1923, -1924, -1925, -1926, and -1927.
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