Translation and post-translational control: ribosomes, chaperones, and protein quality control

Short version. Translation is the decoding of mRNA into protein on the ribosome — an 80S ribonucleoprotein complex of two subunits whose atomic structure was solved at the turn of the millennium and which is, at its catalytic centre, an RNA enzyme. Translation has four stages (initiation, elongation, termination, recycling) and is regulated principally at initiation, through the cap-binding eIF4F complex, the ternary complex eIF2-GTP-Met-tRNAi, the mTOR pathway, and eIF2α phosphorylation in the integrated stress response. After translation, polypeptides are folded by chaperones, modified covalently in many ways, and eventually cleared by the ubiquitin-proteasome system or by autophagy.

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Ribosome structure

The eukaryotic ribosome is an 80S ribonucleoprotein complex with a sedimentation coefficient that reflects the assembly of two subunits:

• Small (40S) subunit: contains the 18S rRNA and around 33 ribosomal proteins. Carries the decoding centre, where the codon-anticodon interaction between mRNA and aminoacyl-tRNA is monitored.
• Large (60S) subunit: contains the 28S, 5.8S, and 5S rRNAs and around 47 ribosomal proteins. Carries the peptidyl transferase centre (PTC), the catalytic site for peptide bond formation, and the polypeptide exit tunnel through which the nascent chain emerges.

The atomic structures of the bacterial ribosome subunits, on which the eukaryotic structures rest, were solved at the turn of the century: the large subunit by Ban, Nissen, Hansen, Moore and Steitz (2000, Science 289:905) at 2.4 Å resolution, and the small subunit by Wimberly, Brodersen, Clemons, Morgan-Warren, Carter, Vonrhein, Hartsch and Ramakrishnan (2000, Nature 407:327) at 3.0 Å. A third structure of the small subunit and a 5.5 Å structure of the entire bacterial 70S ribosome with bound tRNAs and mRNA followed shortly. Venki Ramakrishnan, Tom Steitz, and Ada Yonath shared the 2009 Nobel Prize in Chemistry "for studies of the structure and function of the ribosome".

The structures established two facts that had been hypothesised but not directly demonstrated. First, the peptidyl transferase centre is built entirely of RNA: the catalytic site is rRNA, with no protein side chain within several ångströms of the substrate. The ribosome is a ribozyme, and the most important enzyme of cellular life is an RNA enzyme — striking support for an RNA-world view of the origin of translation. Second, the decoding centre on the small subunit uses two universally conserved adenosines (A1492 and A1493 in the 16S rRNA) to monitor the geometry of the codon-anticodon helix and so distinguish cognate from near-cognate tRNAs.

Initiation, elongation, termination, recycling

Eukaryotic translation has four stages, each with its own factor cohort. The mechanism is reviewed in Jackson, Hellen and Pestova (2010, Nature Reviews Molecular Cell Biology 11:113).

Initiation. The eIF4F cap-binding complex (eIF4E + eIF4G + eIF4A) recognises the 5' cap; eIF4G recruits the 43S pre-initiation complex (40S subunit, eIF1, eIF1A, eIF3, eIF5, and the ternary complex eIF2-GTP-Met-tRNAi). The complex scans the 5' UTR in a 5' to 3' direction, with eIF4A providing the helicase activity that resolves secondary structure. At the start codon (typically AUG in a Kozak context), codon-anticodon pairing triggers eIF1 release, eIF5-stimulated GTP hydrolysis on eIF2, and recruitment of the 60S subunit by eIF5B-GTP. The result is an 80S initiation complex with Met-tRNAi base-paired with the AUG in the P site.

Elongation. The cycle has three stages: (1) eEF1A-GTP delivers an aminoacyl-tRNA to the A site, where the codon-anticodon interaction is monitored; cognate pairing triggers GTP hydrolysis on eEF1A; (2) the peptidyl transferase centre catalyses peptide bond formation, transferring the nascent chain from the P-site tRNA to the A-site aminoacyl-tRNA; (3) eEF2-GTP catalyses translocation, moving the deacylated tRNA from P to E and the peptidyl-tRNA from A to P, ratcheting the small subunit relative to the large.

Termination. A stop codon (UAA, UAG, UGA) in the A site is recognised by eRF1 (eukaryotic release factor 1), which mimics the shape of an aminoacyl-tRNA and triggers hydrolysis of the peptidyl-tRNA in cooperation with eRF3-GTP. The completed polypeptide is released.

Recycling. The post-termination complex is disassembled by ABCE1 and the eIF1 / eIF1A / eIF3 factors, separating the subunits and releasing the deacylated tRNA and the mRNA for re-use.

Cap-dependent vs IRES-mediated initiation

Most cellular mRNAs are translated by the cap-dependent scanning mechanism described above. A subset of cellular and viral mRNAs use internal ribosome entry sites (IRES): structured 5' UTR elements that recruit the small subunit directly, with reduced or no requirement for eIF4F or scanning. Picornaviral IRES elements are the canonical examples; cellular IRES elements operate in a smaller set of stress-response and cell-cycle transcripts. IRES-mediated initiation allows selective translation of specific transcripts when cap-dependent initiation is suppressed — under viral infection, in apoptosis, during the unfolded protein response.

Translation regulation

Acute regulation of translation is dominated by control of initiation. Two regulatory axes account for most of the action, and are reviewed by Sonenberg and Hinnebusch (2009, Cell 136:731):

• eIF2α phosphorylation and the integrated stress response (ISR). Phosphorylation of eIF2α on Ser51 by one of four stress-sensing kinases — PERK (endoplasmic reticulum stress, the unfolded protein response), GCN2 (amino acid starvation, UV), PKR (double-stranded RNA, viral infection), HRI (haem deficiency, oxidative stress) — converts eIF2 from a substrate of its guanine nucleotide exchange factor eIF2B into a competitive inhibitor. The ternary complex eIF2-GTP-Met-tRNAi is depleted, and global cap-dependent initiation is suppressed. A small set of transcripts containing upstream open reading frames (uORFs), notably ATF4 and CHOP in mammals, is paradoxically translationally upregulated under these conditions, driving the ISR transcriptional programme.
• The mTOR pathway and eIF4E availability. The mechanistic Target Of Rapamycin complex 1 (mTORC1) integrates growth-factor, amino-acid, and energy signals. Active mTORC1 phosphorylates the 4E-BP family of eIF4E inhibitors, releasing eIF4E to assemble the eIF4F cap-binding complex; mTORC1 also phosphorylates and activates p70 S6 kinase (S6K), which in turn phosphorylates ribosomal protein S6 and the eIF4B helicase activator. The net effect is upregulation of cap-dependent translation initiation; the effect is particularly strong on transcripts with structured 5' UTRs and on the TOP-mRNAs that encode ribosomal proteins and translation factors.

Additional regulatory layers include codon-usage-mediated control of elongation rate, translational pausing at structured mRNA elements, microRNA-mediated repression, and trans-acting RNA-binding proteins that mask or unmask specific transcripts.

Polysome and ribosome profiling

Polysome profiling separates mRNAs by the number of ribosomes loaded on them, using sucrose density gradient centrifugation. Transcripts in heavy polysomes are inferred to be efficiently translated; transcripts in the monosome or sub-monosome fractions are inferred to be poorly translated. The method gives a transcript-resolution view of translation but no information about which codons the ribosomes are at.

Ribosome profiling (Ribo-seq) takes the picture to codon resolution. Introduced by Ingolia, Ghaemmaghami, Newman and Weissman (2009, Science 324:218), the method works by halting translation in cells (typically with cycloheximide, harringtonine, or flash-freezing), digesting unprotected RNA with nuclease, isolating the ~28-nucleotide mRNA fragments protected by ribosomes, and deep-sequencing them. The resulting density of ribosome footprints across each transcript gives a snapshot of where translating ribosomes are at the moment of harvest, supporting analysis of translation rate, uORF usage, alternative start-site selection, and translation of non-canonical open reading frames. Ribosome profiling has become the dominant high-throughput approach to translation.

Co-translational folding and chaperones

A polypeptide does not wait for translation to finish before it begins to fold. Co-translational folding starts as the nascent chain emerges from the ribosomal exit tunnel; chaperones engage the chain at the tunnel exit and assist folding while elongation continues. The chaperone networks that handle folding, refolding, and triage of misfolded clients are reviewed in Hartl, Bracher and Hayer-Hartl (2011, Nature 475:324):

• Hsp70 (DnaK in bacteria; HSPA family in eukaryotes): an ATP-dependent chaperone that binds short hydrophobic segments exposed in nascent or unfolded clients. J-domain co-chaperones (HSP40 / DNAJ family) deliver clients; nucleotide exchange factors release them. Hsp70 cycles substrates through bound and free states, suppressing aggregation and supporting folding.
• Hsp90 (HSPC family in eukaryotes): an ATP-dependent dimeric chaperone that handles a large set of metastable clients including kinases, transcription factors, and steroid receptors. Hsp90 acts late in folding, after Hsp70.
• GroEL / GroES (HSP60 / HSP10 in mitochondria; CCT / TRiC in the eukaryotic cytosol): chaperonins, double-ring complexes that enclose a single client in a folding chamber capped by the co-chaperonin lid; ATP-driven cycling between open and closed states gives the substrate a confined, hydrophilic environment in which to fold.
• Other systems: small Hsps (sHsps, e.g. HSPB family) act as ATP-independent holdases for stressed clients; the prefoldin complex feeds substrates to the eukaryotic chaperonin; nascent-polypeptide-associated complex (NAC) and the signal recognition particle (SRP) act at the ribosome exit tunnel, handing chains to the cytosolic chaperone network or to the translocon.

Protein quality control: ubiquitin-proteasome and autophagy

Proteins that fail to fold, that are damaged, or that have outlived their usefulness must be cleared. Two systems do most of the work:

• The ubiquitin-proteasome system (UPS). Substrates are tagged with poly-ubiquitin chains by an E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligase) enzyme cascade. Humans have two E1s, around 40 E2s, and over 600 E3s; substrate selectivity is encoded principally in the E3 layer. K48-linked poly-ubiquitin is the canonical degradation signal. The 26S proteasome — a 2.5 MDa complex consisting of a 20S proteolytic core (a barrel of four stacked rings, with the catalytic threonine residues in the inner two rings) capped by 19S regulatory particles — recognises ubiquitinated substrates, recycles the ubiquitin, unfolds the substrate (an ATP-dependent step performed by the AAA+ ATPase ring of the 19S particle), and translocates it into the catalytic chamber for hydrolysis to short peptides.
• Autophagy. Bulk and selective degradation of cytoplasmic contents through delivery to the lysosome / vacuole. Macroautophagy engulfs cargo in a double-membrane autophagosome that fuses with the lysosome; mitophagy, ribophagy, aggrephagy, and other selective autophagy pathways use cargo-receptor proteins (p62 / SQSTM1, NBR1, OPTN, NDP52) to target specific organelles or aggregates. The ATG protein machinery, identified in yeast genetic screens for which the 2016 Nobel Prize was awarded to Yoshinori Ohsumi, builds the autophagosome.

Mis-handling of folding or quality control is a major source of human disease. Cystic fibrosis (mis-folding of CFTR-ΔF508 caught by ER quality control), α1-antitrypsin deficiency (polymerised mis-folded protein in hepatocytes), and many neurodegenerative diseases (aggregation of α-synuclein, tau, huntingtin) sit on this axis.

Post-translational modifications

The polypeptide leaving the ribosome is a starting point, not a finished product. Covalent modifications adjust activity, localisation, interactions, and turnover. The major modifications are:

• Phosphorylation: transfer of a phosphate group to Ser, Thr, or Tyr (also His in some contexts) by a protein kinase; reversed by phosphatases. Around a third of human proteins are phosphorylated; phosphorylation is the dominant covalent mechanism of acute signalling.
• Acetylation: transfer of an acetyl group to lysine (most studied on histones and on metabolic enzymes), catalysed by lysine acetyltransferases (KATs / HATs) and reversed by lysine deacetylases (KDACs / HDACs / sirtuins).
• Methylation: methyl-group transfer to Lys or Arg, catalysed by lysine and arginine methyltransferases; central to the histone code and to the regulation of many non-histone substrates.
• Ubiquitination: covalent attachment of ubiquitin to Lys (or, more rarely, to N-termini, Cys, Ser, or Thr); chain-type-dependent functions span proteasomal degradation (K48), signalling (K63), inflammation (M1 / linear), and many specialised roles.
• Glycosylation: covalent attachment of carbohydrate, with the major classes being N-linked glycosylation on Asn (in the secretory pathway), O-linked glycosylation on Ser / Thr (mucin-type O-GalNAc, O-GlcNAc, and others), GPI anchoring at C-termini, and proteoglycan glycosaminoglycan modification. N-linked glycosylation is essential to the folding and quality control of secreted and membrane proteins.
• SUMOylation: covalent attachment of SUMO (Small Ubiquitin-like MOdifier) to Lys, with effects on transcription factor activity, nuclear-cytoplasmic transport, DNA repair, and chromatin organisation.
• Other modifications: lipidation (myristoylation, palmitoylation, prenylation) anchoring proteins to membranes; ADP-ribosylation by the PARP family in DNA damage response; hydroxylation of proline (in collagen and HIF-α); citrullination by PADs; many more.

The combinatorial layering of these modifications — sometimes called the "PTM code" — expands the functional repertoire of a fixed proteome and links it dynamically to the upstream regulation covered on the transcriptional machinery and RNA processing and stability pages.

Frequently asked questions

Related reading

• Gene expression mechanisms — pillar overview
• Transcriptional machinery
• RNA processing and stability
• Mendelian inheritance calculator