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Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex

ABSTRACT: Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance, and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular detail on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling

SUBMITTER: Dr. Elina Nürenberg-Goloub

PROVIDER: S-SCDT-EMBOJ-2019-103788 | biostudies-other |

REPOSITORIES: biostudies-other

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Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex.

Project description:Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.

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The ribosome-bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex.

Project description:Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet-tRNA(fMet) to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet-tRNA(fMet) to the ribosome in a ternary complex, IF2.GTP.fMet-tRNA(fMet). By using rapid kinetic techniques, we show here that binding of IF2.GTP to the 30S ribosomal subunit precedes and is independent of fMet-tRNA(fMet) binding. The ternary complex formed in solution by IF2.GTP and fMet-tRNA is unstable and dissociates before IF2.GTP and, subsequently, fMet-tRNA(fMet) bind to the 30S subunit. Ribosome-bound IF2 might accelerate the recruitment of fMet-tRNA(fMet) to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF-Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.

| S-EPMC2854590 | biostudies-literature

Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting.

Project description:We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.

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Exploring the flexibility of ribosome recycling factor using molecular dynamics.

Project description:Ribosome recycling factor is proposed to be flexible, and that flexibility is believed to be important to its function. Here we use molecular dynamics to test the flexibility of Escherichia coli RRF (ecRRF) with and without decanoic acid bound to a hydrophobic pocket between domains 1 and 2, and Thermus thermophilus RRF (ttRRF) with and without a mutation in the hinge between domains 1 and 2. Our simulations show that the structure of ecRRF rapidly goes from having an interdomain angle of 124 degrees to an angle of 98 degrees independently of the presence of decanoic acid. The simulations also show that the presence or absence of decanoic acid leads to changes in ecRRF flexibility. Simulations of wild-type and mutant ttRRF (R32G) show that mutating Arg-32 to glycine decreases RRF flexibility. This was unexpected because the range of dihedral angles for arginine is limited relative to glycine. Furthermore, the interdomain angle of wild-type T. thermophilus goes from 81 degrees to 118 degrees whereas the R32G mutant remains very close to the crystallographic angle of 78 degrees . We propose that this difference accounts for the fact that mutant ttRRF complements an RRF deficient strain of E. coli whereas wild-type ttRRF does not. When the ensemble of RRF structures is modeled into the ribosomal crystal structure, a series of overlaps is found that corresponds with regions where conformational changes have been found in the cryoelectron microscopic structure of the RRF/ribosome complex, and in the crystal structure of a cocomplex of RRF with the 50S subunit. There are also overlaps with the P-site, suggesting that RRF flexibility plays a role in removing the deacylated P-site tRNA during termination of translation.

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Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor.

Project description:Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced. The P. aeruginosa frr was mapped at 30 to 32 min of the P. aeruginosa chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of E. coli RRF. In an assay including E. coli polysome and elongation factor G, purified recombinant RRF of P. aeruginosa released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr. The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E. coli and Bacillus subtilis. Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.

| S-EPMC93507 | biostudies-literature

Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling.

Project description:During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling.

| S-EPMC5555083 | biostudies-literature

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry.

Project description:Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final-or the first-step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix-loop-helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling.

| S-EPMC5105147 | biostudies-literature

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome.

Project description:Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

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Structural Insights into ribosome recycling factor interactions with the 70S ribosome.

Project description:At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 A, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.

| S-EPMC2712656 | biostudies-literature

Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth.

Project description:Ribosome releasing factor, product of the frr gene in Escherichia coli, is responsible for dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes and is renamed ribosome recycling factor in this paper. An E. coli strain was constructed (MC1061-2), which carried frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid. MC1061-2 is temperature-sensitive in its growth and does not segregate its frr-carrying plasmid under the plasmid incompatibility pressure. In contrast, isogenic E. coli carrying wild-type frr in the chromosome and mutated frr on the temperature-sensitive plasmid is not temperature-sensitive in its growth and segregates its plasmid from incompatible plasmids. All spontaneously formed thermoresistant colonies derived from MC1061-2 carried wild-type frr that resided either in the bacterial chromosome by re-exchange or in the plasmid, which became temperature-resistant. These observations establish that frr is an essential gene for cell growth.

| S-EPMC43762 | biostudies-other

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