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Review
, 19 (8), 526-541

Roadblocks and Resolutions in Eukaryotic Translation

Affiliations
Review

Roadblocks and Resolutions in Eukaryotic Translation

Anthony P Schuller et al. Nat Rev Mol Cell Biol.

Abstract

During protein synthesis, ribosomes encounter many roadblocks, the outcomes of which are largely determined by substrate availability, amino acid features and reaction kinetics. Prolonged ribosome stalling is likely to be resolved by ribosome rescue or quality control pathways, whereas shorter stalling is likely to be resolved by ongoing productive translation. How ribosome function is affected by such hindrances can therefore have a profound impact on the translational output (yield) of a particular mRNA. In this Review, we focus on these roadblocks and the resumption of normal translation elongation rather than on alternative fates wherein the stalled ribosome triggers degradation of the mRNA and the incomplete protein product. We discuss the fundamental stages of the translation process in eukaryotes, from elongation through ribosome recycling, with particular attention to recent discoveries of the complexity of the genetic code and regulatory elements that control gene expression, including ribosome stalling during elongation, the role of mRNA context in translation termination and mechanisms of ribosome rescue that resemble recycling.

Figures

Figure 1
Figure 1. Overview of eukaryotic translation
(A) Overview of eukaryotic translation. Translation begins with initiation where a complex coordination of many eukaryotic translation initiation factors (eIFs), initiator methionyl-tRNA (Met-tRNAiMet), the ribosomal subunits and mRNA to be translated come together at the AUG start codon of the open reading frame. Next, elongation involves synthesis of the peptide chain through the coordinated actions of eukaryotic elongation factors (eEFs) and aminoacyl-tRNAs (aa-tRNAs) until the ribosome reaches a termination or stop codon. In this termination phase the peptide is released through the actions of eukaryotic peptide chain release factors (eRFs). Finally, the ribosome subunits must be recycled by ATP-binding cassette sub-family E member 1 (ABCE1) for a subsequent round of translation. (B) Structural model of the yeast 80S ribosome depicting three sites for tRNA binding: E (exit; colored orange), P (peptidyl; colored purple) and A (aminoacyl; colored cyan). mRNA is shown in red. The model was created by alignment of the Saccharomyces cerevisiae 80S ribosome (PDB 4V88) and Thermus thermophilus 70S ribosome (PDB 4V9I). (C) Overview of the elongation cycle highlighting tRNA movement through the ribosome. tRNA selection occurs at the A site. Next, peptide-bond formation occurs, which transfers the peptide to the A-site tRNA. Concurrent with peptide bond formation, the tRNAs adopt a ‘hybrid’ state (relative to the large and small ribosomal subunits) and this ribosome complex is the substrate for translocation to allow the decoding of the next codon. (D) Schematic of the peptidyl-transfer reaction that occurs during translation elongation. The amino group of the incoming amino acid (cyan) attacks the ester linkage on the peptidyl-tRNA (purple) in the ribosomal P site to transfer the growing peptide chain to A-site tRNA. (E) Structural model of the S. cerevisiae 80S ribosome bound to eIF5A. eIF5A binds in the ribosomal E site. Zoom-in area shows interactions between the hypusine modification of eIF5A and the CCA nucleotides at the 3′-end of the peptidyl-tRNA. The model was created by alignment of the S. cerevisiae 80S ribosome (PDB 4V88) with coordinates for the 60S subunit bound by eIF5A (PDB 5GAK).
Figure 2
Figure 2. Translation elongation and resolution of ribosome stalling
(A) Overview of translation elongation. Aminoacyl-tRNAs are delivered to the ribosome in complex with elongation factor 1-alpha (eEF1A) and GTP (not shown). Peptide bond formation occurs and the tRNAs are positioned in a ‘hybrid’ state with respect to the ribosome subunits. Subsequent translocation driven by elongation factor 2 (eEF2) causes tRNA repositioning from a hybrid state to classical state, creating an open A site for the next incoming aminoacyl-tRNA. eIF5A, which is a small protein that binds in the ribosomal E site, stimulates catalysis in the peptidyl transferase center throughout translation elongation. (B) Ribosome stalling due to slow peptidyl-transfer kinetics (such as during the formation of Pro–Pro) is rescued by eIF5A, which promotes peptide-bond formation. (C) Ribosome stalling caused by poor A-site occupancy resulting from poorly-expressed tRNAs (represented in figure) or poor tRNA aminoacylation can be rescued by mis-incorporation of near-cognate tRNAs or by frameshifting (represented as conversion of orange to purple). (D) Ribosome stalling can be caused by certain consecutive tRNA–codon pair orders that are sub-optimal (pink and orange) relative to synonymous pairs (purple and blue). (E) Ribosome stalling caused by mRNA secondary structures can be resolved by programmed ribosomal frameshifting (PRF) at adjacent slippery sequences. The example illustrates the -1 PRF that is required for translation of the Gag-Pol fusion protein of human immunodeficiency virus.
Figure 3
Figure 3. Translation termination and the role of mRNP context
(A) Overview of translation termination. When the ribosome encounters a termination (stop) codon, eukaryotic peptide chain release factor 1 (eRF1) is delivered by eukaryotic peptide chain release factor 3 (eRF3) to catalyze peptidyl-hydrolysis at the ribosome peptidyl-transferase center. eIF5A binds in the ribosomal E site to stimulate eRF1-mediated hydrolysis. (B) Structure of the 80S–eRF1–ATP-binding cassette sub-family E member 1 (ABCE1) complex (PDB 3JAH) with close-up view showing the GGQ motif of eRF1, which is positioned to coordinate a water molecule for peptidyl-hydrolysis of the P-site peptidyl-tRNA. (C) Schematic of peptidyl-release reaction coordinated by eRF1. (D) Proximity to poly(A)-binding protein (PABP) and other stimulatory RNA-binding proteins (RBPs) can affect translation termination efficiency. (E) Alternatively, if a termination codon is located in a non-ideal mRNP context, far from PABP or stimulatory RBPs, or if certain inhibitory RBPs (colored red) are present near the stop codon, the ribosome may terminate inefficiently. In these cases, the ribosome may undergo frameshifting or incorporate a near-cognate tRNA (orange) to continue translation until a more ideal stop codon is reached.
Figure 4
Figure 4. Ribosome recycling and rescue
(A) Overview of ribosome recycling by ABCE1. ATP-binding cassette sub-family E member 1 (ABCE1) binds to 80S ribosomes loaded with eukaryotic peptide chain release factor subunit 1 (eRF1) and uses the power generated from ATP-binding and hydrolysis to dissociate the ribosomal subunits. ABCE1 remains bound to the 40S subunit to stimulate subsequent translation initiation steps. (B) Superposition of ABCE1 structures in the pre-splitting (PDB 3JAH) and post-splitting (PDB 5LL6) states to highlight the 150-degree rotation of the iron-sulfur (Fe–S) cluster that occurs during ribosomal subunit dissociation. (C) Superposition of ABCE1 structures pre- and post-splitting in the context of eRF1 and ribosomal protein uL14. In the pre-splitting state, the Fe–S cluster of ABCE1 interacts with eRF1. Following splitting, the Fe–S cluster undergoes a dramatic conformational change which drives eRF1 into the inter-subunit space of the ribosome to promote subunit dissociation. In the post-splitting state the Fe–S cluster would clash with 60S ribosomal protein uL14, thereby preventing 60S rejoining and effectively completing the recycling reaction. (D) Dom34–Hbs1 in coordination with ABCE1 rescues stalled ribosomes at the truncated 3′-ends of mRNAs resulting from endo- or exo-nucleolytic cleavage, or those found translating the poly(A) tail. (E) Inactive or ‘hibernating’ Stm1-bound 80S ribosomes in Saccharomyces cerevisiae can be rescued by Dom34–Hbs1 in coordination with ABCE1 to re-enter the cytoplasmic pool of translating ribosomes.

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