Tightly-orchestrated rearrangements govern catalytic center assembly of the ribosome

Abstract

The catalytic activity of the ribosome is mediated by RNA, yet proteins are essential for the function of the peptidyl transferase center (PTC). In eukaryotes, final assembly of the PTC occurs in the cytoplasm by insertion of the ribosomal protein Rpl10 (uL16). We determine structures of six intermediates in late nuclear and cytoplasmic maturation of the large subunit that reveal a tightly-choreographed sequence of protein and RNA rearrangements controlling the insertion of Rpl10. We also determine the structure of the biogenesis factor Yvh1 and show how it promotes assembly of the P stalk, a critical element for recruitment of GTPases that drive translation. Together, our structures provide a blueprint for final assembly of a functional ribosome.

Fig. 1

Structures of late nuclear and cytoplasmic particles reveal the pathway of 60S maturation. Crown view of late nuclear (LN) (a), early cytoplasmic-immediate (ECI) (b), early cytoplasmic-late (ECL) (c), pre-Lsg1 (PL) (d), Lsg1-engaged (LE) (e), and Rpl10 (uL16)-inserted (RI) (f) ribosomal intermediates at ~3.5–3.8 Å resolution, with corresponding atomic models shown on the right. Key protein and ribosomal RNA (rRNA) elements are colored as indicated. Black arrows indicate conformational changes while gray arrows indicate protein exchanges. CP, central protuberance. In the early cytoplasmic-immediate (ECI) particle, Rpl12 (uL11) was not modeled due to poor density. In the early cytoplasmic-late (ECL) particle, Rpl12 is not clearly discernible and Mrt4 is poorly resolved due to the mobility of the P stalk after the N-terminal domain (NTD) of Nog1 is released from the A site. Therefore, Rpl12 was not modeled, and Mrt4 was only partially modeled

Fig. 2

Yvh1 forces the release of Mrt4. a Atomic structure of the zinc-binding domain of Yvh1 modeled into the unassigned density. The small piece of extra density (blue) was assigned to the C terminus of Tif6 (see Supplementary Fig. 7b). b–d Interactions of Yvh1 with helices H43 and H44 (b), helix H89 (c), and sarcin-ricin loop (SRL) (d). Dashed lines, predicted hydrogen bonds. e Comparison of the P stalk RNAs in the Mrt4-bound (orange) and Yvh1-bound (blue) states. f Mrt4 in cartoon and transparent surface representation. A1221 of H42 bound in a pocket of Mrt4. g Mrt4 bound to P stalk RNA rotated ~120° clockwise relative to the view in (a) showing A1221 of H42 in the binding pocket of Mrt4. Residues of Mrt4 interacting with H42 are indicated. h Mrt4 from the early cytoplasmic-immediate (ECI) particle was docked onto H43 and H44 of the Yvh1-bound pre-Lsg1 (PL) particle, showing the movement of Mrt4 away from A1221 of H42

Fig. 3

Large-scale rearrangements of Nmd3 N-terminal domain (NTD) primes the subunit for Rpl10 (uL16) insertion and completion of the catalytic center. a Atomic structure of the zinc-binding NTD of Nmd3. Cysteines of the two treble clef zinc-binding motifs are indicated. Zn²⁺ ions were modeled into their predicted positions. b Interactions of H69 with Nmd3 and Lsg1 showing flipped-out G2261 and U2269. c In the pre-Lsg1 particle, H108 of the Nmd3 histidine thumb is engaged with H89. d Upon Lsg1 binding, the bulk of the NTD of Nmd3 rotates ~60°, releasing the histidine thumb from H89. e, f Upon Rpl10 insertion, the middle portion of H89 is retracted ~10 Å to accommodate and stabilize Rpl10 in its binding cleft; the tip of H89 remains in position to interact with Yvh1

Fig. 4

Release of H38 and H89 from Nmd3 stabilizes Rpl10 (uL16) in the ribosome. a Atomic structure showing that H38 lays in a saddle of Nmd3 (top). Lower panel, selected residues highlighted in orange sit in the immediate interface between Nmd3 and H38. L291, N332, and I362 (purple) were previously identified from genetic screens for suppressors the temperature-sensitive rpl10-G161D mutant. b Atomic structure showing that interaction of the histidine thumb of Nmd3 with H89. H108 and I112 were identified from genetic screens rpl10-G161D suppressors. c Wild-type (WT) or rpl10-G161D mutant cells were transformed with empty vector or vector expressing WT or the indicated NMD3 mutants. Ten-fold serial dilutions of cultures were plated onto selective plates and incubated for 2 days at 30 °C, a semi-permissive temperature for rpl10-G161D. The mutations H167A, R169A, R333A, K400A, and Y402A in NMD3 were engineered based on the structural information as indicated in (a)

Fig. 5

Summary of 60S maturation concluding with Rpl10 (uL16) insertion. a Late nuclear particle before Nmd3 loading. H38 tip is flexible while H89 is displaced by the N-terminal domain (NTD) of Nog1 in the A site. Rpl12 (uL11) and Mrt4 both bind to the P stalk. b Early cytoplasmic-immediate particle showing that the nuclear export factor Nmd3 closes the L1 stalk and captures H38. The NTD of Nog1 remains in the A site, displacing H89, and the NTD of Nmd3 is not discernible, probably because of its high mobility before docking onto Tif6. c In the early cytoplasmic-late particle, the NTD and GTPase domain of Nog1 have been released from the A site, allowing Nmd3 to dock on Tif6 and bind H89, which is rearranged to its near-mature position. Note that the Nog1 C terminus remains in place while the NTD and GTPase domain are not discernible, likely due to high flexibility after displacement from the A site. d In the pre-Lsg1 particle, multiple assembly factors have been released. The release of Nog1 allows the binding of Yvh1 to release Mrt4 from the P stalk. e The Lsg1-engaged particle reveals a rotation of the NTD of Nmd3 away from H89 to engage with Lsg1. This releases H89 from Nmd3 to prime the subunit for the insertion of Rpl10. f The insertion of Rpl10 causes retraction of both H38 and H89 to their mature positions to stabilize Rpl10 in its binding site. The complete release of Nmd3 from H38 and H89 poises Nmd3 for its imminent release. Subsequently, the nascent 60S subunit undergoes a test drive using molecular mimics of translation factors before licensing it for bona fide translation