Biochemistry

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Translation (Protein Synthesis) - A Complete Guide

Translation is the process by which the nucleotide sequence encoded in mRNA is read by the ribosome and converted into a sequence of amino acids - a polypeptide chain. It takes place in the cytoplasm and has three main phases: Initiation, Elongation, and Termination.

Prerequisites: Key Molecules

Before translation can begin, three types of RNA and several accessory proteins must be in place:

Molecule	Role
mRNA	Carries the genetic message (codons, read 5'→3')
tRNA	Adaptor molecules; each carries a specific amino acid and has an anticodon that pairs with the mRNA codon
rRNA	Structural and catalytic component of the ribosome
Aminoacyl-tRNA synthetases	Enzymes that attach ("charge") the correct amino acid to the correct tRNA, consuming 2 ATP equivalents per amino acid

Charging tRNA: Each amino acid is first activated by reacting with ATP (forming aminoacyl-AMP), then transferred to the 3'-OH end of its specific tRNA. Recognition of the correct tRNA by its synthetase uses the anticodon sequence (and sometimes other positions on the tRNA molecule).

Basic Medical Biochemistry, 6e, p. 480

Ribosomes

Prokaryotes: 70S ribosome = 30S (small) + 50S (large) subunits
Eukaryotes: 80S ribosome = 40S (small) + 60S (large) subunits

The ribosome has three tRNA binding sites:

A site (aminoacyl) - where new aminoacyl-tRNA enters
P site (peptidyl) - holds the tRNA attached to the growing chain
E site (exit) - where the deacylated (empty) tRNA leaves

Phase 1: Initiation

The goal is to assemble the complete ribosome at the AUG start codon of the mRNA.

In Eukaryotes:

The initiator methionyl-tRNA (Met-tRNA$_{Met}$) forms a ternary complex with eIF2-GTP
This complex binds the small 40S subunit (along with eIF3, which prevents premature 60S joining) - forming the 43S preinitiation complex
The 5' cap of the mRNA is recognized by the eIF4F cap-binding complex (eIF4E + eIF4A + eIF4G)
The mRNA-eIF4F complex joins the 43S complex, forming the 48S initiation complex
The ribosome scans the mRNA from the 5' cap toward the 3' end (using eIF4A helicase activity + ATP) until it reaches the AUG codon - guided by the Kozak consensus sequence (purine-CCAUGG)
GTP is hydrolyzed, all initiation factors are released, and the 60S subunit joins - forming the complete 80S ribosome
Met-tRNA$_{Met}$ occupies the P site, and the A site is empty, ready for elongation

In Prokaryotes (key differences):

Initiator tRNA is formyl-methionyl-tRNA (fMet-tRNA)
Only 3 initiation factors needed (IF1, IF2, IF3)
AUG is located by the Shine-Dalgarno sequence in mRNA, which base-pairs with the 3' end of 16S rRNA in the 30S subunit (no scanning)
Basic Medical Biochemistry, 6e, pp. 481-482
Harper's Illustrated Biochemistry, 32e, p. 421

Phase 2: Elongation

Each amino acid addition is a 3-step cycle, repeated for every residue. Each cycle consumes 2 GTP.

Overview of translation elongation showing the P, A, and E sites of the ribosome, with tRNAs moving through as the polypeptide chain grows

Step 1 - Aminoacyl-tRNA Binding at the A Site

eEF1A-GTP (EF-Tu in prokaryotes) forms a ternary complex with the incoming aminoacyl-tRNA
This complex delivers the correct aminoacyl-tRNA to the A site - correct codon-anticodon pairing triggers GTP hydrolysis
eEF1A-GDP is released and recycled back to eEF1A-GTP

Step 2 - Peptide Bond Formation

Peptidyl transferase (catalytic activity of the large rRNA - a ribozyme) transfers the growing polypeptide from the P-site tRNA onto the amino acid on the A-site tRNA
The result: a peptidyl-tRNA (one residue longer) at the A site, and a deacylated tRNA at the P site

Step 3 - Translocation

eEF2-GTP (EF-G in prokaryotes) binds the ribosome, causing a conformational change
The ribosome shifts 3 nucleotides (one codon) in the 5'→3' direction:
- Deacylated tRNA moves P → E site (and is ejected when the next aminoacyl-tRNA enters)
- Peptidyl-tRNA moves A → P site
- A site is now empty, displaying the next codon
GTP is hydrolyzed to GDP + Pi and eEF2 is released

mRNA being read 5' to 3' with outgoing and incoming tRNAs exchanging at the ribosome during each elongation cycle

This cycle repeats until a stop codon is encountered.

Basic Medical Biochemistry, 6e, pp. 484-486

Phase 3: Termination

When a stop codon (UAA, UAG, or UGA) enters the A site, no tRNA with a matching anticodon exists
Release factors (eRF1 and eRF3 in eukaryotes; RF1, RF2, RF3 in prokaryotes) bind to the ribosome at the stop codon
Peptidyl transferase hydrolyzes the bond between the polypeptide chain and the P-site tRNA - releasing the completed protein
The ribosome dissociates into its 40S + 60S subunits, releasing the mRNA
Subunits are recycled for the next round of translation
Basic Medical Biochemistry, 6e, p. 486

Energy Cost

Step	Energy Used
Amino acid activation (tRNA charging)	2 ATP equiv (ATP → AMP + PPi) per amino acid
Aminoacyl-tRNA binding (A site)	1 GTP per amino acid
Translocation	1 GTP per amino acid
Total	4 high-energy bonds per amino acid added

Basic Medical Biochemistry, 6e, p. 486

Polysomes

Once one ribosome moves away from the 5' end of the mRNA, a second ribosome can attach. Multiple ribosomes translate the same mRNA simultaneously, forming a polysome (polyribosome). A single ribosome covers ~80 nucleotides, so ribosomes are spaced ~100 nucleotides apart. This dramatically increases the rate of protein production from a single mRNA molecule.

Prokaryote vs. Eukaryote Comparison

Feature	Prokaryote	Eukaryote
Ribosome	70S (30S + 50S)	80S (40S + 60S)
Initiator tRNA	fMet-tRNA	Met-tRNA$_{Met}$
Start codon ID	Shine-Dalgarno sequence	5' cap scanning + Kozak sequence
Initiation factors	3 (IF1, IF2, IF3)	10+ (eIFs)
Elongation factor (A site)	EF-Tu	eEF1A
Translocation factor	EF-G	eEF2
Coupled transcription/translation	Yes	No (compartmentalized)

Clinically Relevant Points

Antibiotics exploit ribosome differences between prokaryotes and eukaryotes:
- Tetracyclines - block aminoacyl-tRNA binding to the 30S A site
- Aminoglycosides (streptomycin) - cause mRNA misreading at the 30S subunit
- Chloramphenicol - inhibits peptidyl transferase on the 50S subunit
- Linezolid - prevents formation of the 70S initiation complex (23S rRNA)
- Macrolides (erythromycin) - block the polypeptide exit tunnel in the 50S
Diphtheria toxin: The A subunit of Corynebacterium diphtheriae toxin ADP-ribosylates eEF2 (at a modified histidine called diphthamide), permanently inactivating it and halting protein synthesis - leading to cell death.
eIF2 regulation: Phosphorylation of eIF2 inactivates it, halting translation. This occurs during starvation, heat shock, and viral infection. In reticulocytes, heme inhibits eIF2 phosphorylation, promoting globin synthesis only when heme is available.
Basic Medical Biochemistry, 6e, pp. 482, 486
Harper's Illustrated Biochemistry, 32e, p. 421
Histology: A Text and Atlas, p. 175

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