Translation in eukaryotic cells

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Translation in Eukaryotic Cells

Translation is the process by which the nucleotide sequence of an mRNA is decoded into the amino acid sequence of a protein. In eukaryotes, translation occurs in the cytoplasm on 80S ribosomes and is divided into three main stages: initiation, elongation, and termination, followed by post-translational modifications.

Components Required

Before translation begins, the following must be present:

80S ribosomes (40S small subunit + 60S large subunit), each containing three tRNA-binding sites: A (aminoacyl), P (peptidyl), and E (exit)
mRNA (monocistronic in eukaryotes - one coding region per mRNA, unlike prokaryotes)
Aminoacyl-tRNAs - tRNAs charged with their specific amino acids by aminoacyl-tRNA synthetases
Eukaryotic initiation factors (eIFs), elongation factors (eEFs), and release factors (eRF)
GTP and ATP as energy sources

The Genetic Code & tRNA

Each codon is a three-nucleotide sequence on mRNA read 5'→3'. Of 64 possible codons, 61 specify amino acids and 3 are stop codons (UAA, UAG, UGA). The code is degenerate (multiple codons can specify the same amino acid).

tRNA links codons to amino acids. The anticodon region (7 nucleotides) base-pairs with the mRNA codon in an antiparallel fashion. The wobble hypothesis explains how the 5' nucleotide of the anticodon can form non-Watson-Crick base pairs with the 3' base of the codon, allowing a single tRNA species to recognize more than one codon.

Recognition of the codon by the anticodon. Phenylalanyl-tRNA showing the anticodon arm pairing with UUU codon on mRNA.

Aminoacyl-tRNA synthetases catalyze tRNA charging in a two-step reaction: (1) amino acid activation with ATP to form aminoacyl-AMP-enzyme complex, then (2) transfer of the amino acid to the 3'-OH of the tRNA. The error rate is extremely low (< 1 in 10^6 events). - Harper's Illustrated Biochemistry, 32nd Ed.

Stage 1: Initiation

Initiation assembles the full ribosome on the start codon. It involves at least 10 eukaryotic initiation factors (eIFs) and requires both GTP and ATP.

Step 1 - Ribosomal Dissociation: The 80S ribosome dissociates into 40S and 60S subunits at the end of each translation cycle. eIF-3, eIF-1, and eIF-1A bind the 40S subunit and prevent premature reassociation with the 60S subunit. - Harper's Illustrated Biochemistry, 32nd Ed.

Step 2 - 43S Preinitiation Complex: eIF-2 binds GTP, then recruits the initiator methionyl-tRNAi (Met-tRNAi) - a special tRNA used only at the start codon AUG (distinct from the tRNA used at internal AUG codons). This ternary complex (eIF-2•GTP•Met-tRNAi) joins the 40S subunit to form the 43S preinitiation complex, stabilized by eIF-3, eIF-1A, and eIF-5.

Step 3 - mRNA Binding and Scanning (48S complex): Unlike prokaryotes, eukaryotes lack a Shine-Dalgarno sequence. Instead, the small subunit (aided by eIF-4 family proteins) binds near the 5' cap structure (m7G cap) of the mRNA and scans 5'→3' until it encounters the first AUG codon. This scanning requires ATP hydrolysis. Interactions between cap-binding eIF-4 proteins and poly-A tail-binding proteins cause mRNA circularization, likely preventing use of incompletely processed mRNA. - Biochemistry, Lippincott 8th Ed.

Cap-independent initiation can also occur via binding of the 40S subunit to an Internal Ribosome Entry Site (IRES) near the start codon.

Step 4 - 80S Initiation Complex: Once the AUG is located, the 60S subunit joins the 48S complex (aided by eIF-5 and eIF-5B with GTP hydrolysis) to form the 80S initiation complex. The initiator Met-tRNAi occupies the P site (not the A site, unlike all subsequent aminoacyl-tRNAs). The A site is now empty and ready to accept the next aminoacyl-tRNA.

Key eukaryotic vs. prokaryotic differences: Eukaryotes use methionine as the initiating amino acid (prokaryotes use N-formylmethionine). The initiating codon is AUG in both.

Stage 2: Elongation

Elongation adds amino acids one at a time to the carboxyl end of the growing peptide. Each cycle has three steps:

1. Decoding (Aminoacyl-tRNA delivery to the A site): The aminoacyl-tRNA matching the next codon in the A site is delivered by eEF-1α•GTP (equivalent to prokaryotic EF-Tu•GTP). When the correct anticodon base-pairs with the mRNA codon, GTP is hydrolyzed, eEF-1α•GDP is released, and the aminoacyl-tRNA is accommodated into the A site. The exchange factor eEF-1γ (EF-Ts analog) regenerates eEF-1α•GTP.

2. Peptide Bond Formation (Transpeptidation): Peptidyl transferase - a ribozyme activity intrinsic to the 28S rRNA of the 60S subunit - catalyzes peptide bond formation between the α-carboxyl group of the amino acid in the P site and the α-amino group of the aminoacyl-tRNA in the A site. The growing peptide chain is transferred from the P-site tRNA to the A-site tRNA (transpeptidation), leaving an uncharged tRNA in the P site.

3. Translocation: The ribosome moves exactly 3 nucleotides (one codon) toward the 3' end of the mRNA, facilitated by eEF-2•GTP (equivalent to prokaryotic EF-G) with GTP hydrolysis. This moves:

The uncharged tRNA from P site → E site (then released)
The peptidyl-tRNA from A site → P site
The next codon into the A site

The cycle repeats until a stop codon enters the A site. Because mRNAs are long, many ribosomes can simultaneously translate a single mRNA, forming a polysome (polyribosome). - Biochemistry, Lippincott 8th Ed.

Stage 3: Termination

When one of the three stop codons (UAA, UAG, or UGA) moves into the A site, no aminoacyl-tRNA recognizes it. Instead:

eRF-1 (a single eukaryotic release factor) recognizes all three stop codons
eRF-1 triggers hydrolysis of the bond between the completed polypeptide and the P-site tRNA, releasing the protein
eRF-3•GTP (analogous to prokaryotic RF-3) functions with eRF-1 and assists in release of eRF-1 upon GTP hydrolysis
The ribosomal subunits, mRNA, and tRNA are released and recycled (requiring eRF and ATP hydrolysis in eukaryotes)

In prokaryotes, two release factors (RF-1 and RF-2) are needed; RF-1 recognizes UAA and UAG, RF-2 recognizes UGA and UAA.

Regulation of Translation

eIF-2 Phosphorylation (Key Control Point)

Under cellular stress conditions (amino acid starvation, heme deficiency, misfolded proteins in RER, viral infection with double-stranded RNA), specific kinases phosphorylate eIF-2α at Serine 51:

Kinase	Trigger
HCR	Heme deficiency
PKR	Viral dsRNA
PERK	ER stress / misfolded proteins
GCN2	Amino acid / glucose starvation

Phosphorylated eIF-2α binds tightly to eIF-2B (the GEF that recycles GDP→GTP on eIF-2), inactivating it. This prevents formation of the 43S preinitiation complex and globally shuts down protein synthesis. This is a protective mechanism - PKR in particular provides antiviral defense by halting viral protein synthesis.

Regulation of translation initiation by phosphorylation of eIF-2. Stress signals activate kinases that phosphorylate eIF-2α, inactivating eIF-2B and blocking translation.

mTOR and eIF-4E-BP1

The mTOR pathway also regulates translation initiation. When active, mTOR phosphorylates 4E-BP1 (eIF-4E-binding protein), causing it to release eIF-4E. Free eIF-4E can then bind the 5' cap and initiate translation. When mTOR is inactive, 4E-BP1 sequesters eIF-4E, blocking translation of specific mRNAs.

Post-Translational Processing

After translation, proteins may undergo:

Cleavage - removal of signal sequences or propeptides
Covalent modifications - phosphorylation, glycosylation, acetylation, hydroxylation, etc.
Folding - assisted by chaperone proteins
Targeting - signal sequences direct proteins to organelles or for secretion

Summary Comparison: Eukaryotes vs. Prokaryotes

Feature	Eukaryotes	Prokaryotes
Ribosome	80S (40S + 60S)	70S (30S + 50S)
mRNA type	Monocistronic	Polycistronic
Start amino acid	Methionine	N-formylmethionine
AUG recognition	5' cap + scanning	Shine-Dalgarno sequence
Initiation factors	≥10 eIFs (complex)	3 IFs (simpler)
Elongation factors	eEF-1α, eEF-1γ, eEF-2	EF-Tu, EF-Ts, EF-G
Release factors	eRF-1 (recognizes all 3 stops)	RF-1, RF-2 (separate)
Coupling of transcription/translation	No (nuclear membrane separates them)	Yes (simultaneous)
Energy for initiation	GTP + ATP	GTP

Sources: Biochemistry, Lippincott Illustrated Reviews 8th Ed., pp. 1254-1270; Harper's Illustrated Biochemistry 32nd Ed., pp. 418-440

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