What do the A, P and E sites of the ribosome do?

The A site accepts each incoming aminoacyl-tRNA, the P site holds the peptidyl-tRNA carrying the growing chain, and the E site holds the empty tRNA as it exits. Remember it as Going APE. During each cycle the chain is transferred from the P-site tRNA to the amino acid on the A-site tRNA, then translocation moves the ribosome three nucleotides so the peptidyl-tRNA shifts to the P site and the empty tRNA moves to the E site.

Where does the initiator tRNA bind during translation?

The initiator tRNA binds directly in the P site, the only tRNA that does not enter through the A site. In bacteria it carries N-formylmethionine and is placed by IF-2 bound to GTP after the Shine-Dalgarno sequence aligns the mRNA with 16S rRNA. In eukaryotes it carries methionine and is delivered by eIF-2 bound to GTP to the 40S subunit, which binds the 5 prime cap and scans to the AUG start codon.

What happens at a stop codon during translation?

When UAA, UAG or UGA reaches the A site, no tRNA can pair with it. A release factor binds instead: RF-1 recognizes UAA or UAG and RF-2 recognizes UAA or UGA in bacteria, while a single eRF-1 recognizes all three in eukaryotes. The release factor stimulates hydrolysis of the bond between the P-site tRNA and the polypeptide, which frees the finished protein. A nonsense mutation creates a premature stop codon and a truncated protein.

What is the difference between eIF-2 and eEF-2?

eIF-2 is a eukaryotic initiation factor: bound to GTP, it carries methionyl initiator tRNA to the 40S subunit, and phosphorylation of eIF-2 shuts down initiation, for example when heme is lacking. eEF-2 is a eukaryotic elongation factor that uses GTP to translocate the ribosome one codon. Diphtheria toxin and Pseudomonas exotoxin A inactivate eEF-2 by ADP-ribosylation, so a stem about stalled translocation points to eEF-2, not eIF-2.

Which antibiotics inhibit the 30S and 50S ribosomal subunits?

Use Buy AT 30, CCEL at 50. Aminoglycosides and tetracyclines bind the 30S subunit: aminoglycosides cause codon-anticodon mismatches and faulty proteins, and tetracyclines block aminoacyl-tRNA entry into the A site. Chloramphenicol, clindamycin, erythromycin and other macrolides, and linezolid bind the 50S subunit: chloramphenicol blocks peptide-bond formation, linezolid blocks formation of the initiation complex, and macrolides and clindamycin are classically said to block translocation, although current texts describe blocked peptide-bond formation.

Translation: Initiation, Elongation & Termination Mnemonic

Q: What are the steps of translation?

Translation has three phases. In initiation, the small ribosomal subunit finds the AUG start codon and the initiator tRNA enters the P site, then the large subunit joins. In elongation, each aminoacyl-tRNA enters the A site, large-subunit rRNA forms the peptide bond, and the ribosome translocates one codon. In termination, a release factor recognizes a stop codon in the A site and the finished chain is released. A memory aid is I-E-T: Invite, Extend, Terminate.

Translation is the ribosome reading mRNA 5′ → 3′ and building a protein from the N-terminus to the C-terminus in three phases. In initiation, the small subunit finds the AUG start codon and the initiator tRNA (formyl-methionine in bacteria, methionine in eukaryotes) goes straight into the P site; in elongation, each aminoacyl-tRNA enters the A site, large-subunit rRNA forms the peptide bond and the ribosome moves one codon; in termination, a release factor, not a tRNA, reads UAA, UAG or UGA in the A site and the finished chain is hydrolyzed off. Charging tRNA costs ATP, while delivering the initiator tRNA, delivering each aminoacyl-tRNA and translocation run on GTP. The classic trap: eIF-2 (initiation) is not eEF-2 (elongation), the factor that diphtheria toxin and Pseudomonas exotoxin A ADP-ribosylate.

The acronym trick: I-E-T, then Going APE

I-E-T = Invite, Extend, Terminate.

Invite (initiation): invite the initiator tRNA into the P site and set the reading frame.
Extend (elongation): extend the chain one codon at a time as tRNAs move A → P → E.
Terminate (termination): a stop sign in the A site brings a release factor, which frees the chain.

Going APE gives the three tRNA sites in the order a tRNA uses them:

Site	Mnemonic	What sits there
A	Aminoacyl (arrival)	Each incoming aminoacyl-tRNA; at a stop codon, the release factor
P	Peptidyl	The tRNA holding the growing chain; at the start, the initiator tRNA
E	Exit	The empty (uncharged) tRNA on its way out

Four more one-liners:

Even eukaryotes, odd prokaryotes: 40S + 60S = 80S; 30S + 50S = 70S.
ATP charges, GTP moves: aminoacyl-tRNA synthetases use ATP (through an aminoacyl-AMP intermediate) to attach each amino acid to the 3′ end of its tRNA; IF-2/eIF-2, EF-Tu/eEF-1α and EF-G/eEF-2 all use GTP.
Stops: "U Are Away, U Are Gone, U Go Away" = UAA, UAG, UGA. Of the 64 codons, 61 encode amino acids.
"Buy AT 30, CCEL at 50": Aminoglycosides and Tetracyclines bind the 30S subunit; Chloramphenicol, Clindamycin, Erythromycin (macrolides) and Linezolid bind the 50S.

Phase by phase: bacteria vs eukaryotes

Step	Bacteria (70S = 30S + 50S)	Eukaryotes (80S = 40S + 60S)
Finding the start	Shine-Dalgarno sequence pairs with the 3′ end of 16S rRNA; can start at internal sites of polycistronic mRNA	eIF-4E binds the 5′ 7-methylguanosine cap; the 40S subunit scans to the AUG
Initiator tRNA	fMet-tRNA, recognized by IF-2-GTP (with IF-1 and IF-3)	Met-tRNAi, carried by eIF-2-GTP
Large subunit joins	50S joins; initiator tRNA sits in the P site	At the AUG, eIF-5 triggers GTP hydrolysis on eIF-2, the factors leave, 60S joins
Aminoacyl-tRNA to the A site	EF-Tu-GTP (recycled by EF-Ts)	eEF-1α-GTP (recycled by eEF-1βγ)
Peptide bond	Peptidyl transferase = large-subunit rRNA (23S)	Peptidyl transferase = large-subunit (60S) rRNA
Translocation	EF-G-GTP	eEF-2-GTP
Stop codon	RF-1 (UAA, UAG), RF-2 (UAA, UGA)	eRF-1 reads all three

Initiation (Invite)

Bacteria: the 30S subunit, IF-1, IF-2 and IF-3 assemble on the mRNA; the Shine-Dalgarno sequence lines up the AUG; IF-2-GTP places fMet-tRNA in the P site, and the 50S subunit joins.
Eukaryotes: eIF-4E recognizes the 5′ cap (added during RNA processing); eIF-2-GTP carries Met-tRNAi to the 40S subunit, which scans to the AUG; eIF-5 triggers GTP hydrolysis, the initiation factors are released and the 60S subunit joins.
Only the initiator tRNA enters the P site directly. Every other aminoacyl-tRNA enters through the A site.
eIF-2 is a control point. In the absence of heme, a protein kinase that phosphorylates eIF-2 is activated and further translation is inhibited, the mechanism behind the practice question below.

Elongation (Extend)

Codon recognition: EF-Tu-GTP (eEF-1α-GTP) brings the matching aminoacyl-tRNA into the A site.
Peptide bond formation: the peptidyl transferase activity of large-subunit rRNA moves the growing chain from the P-site tRNA onto the amino acid on the A-site tRNA. The large subunit still makes peptide bonds after about 95% of its proteins are stripped away: the ribosome is a ribozyme.
Translocation: EF-G-GTP (eEF-2-GTP) moves the ribosome 3 nucleotides along the mRNA. The peptidyl-tRNA shifts A → P, the empty tRNA shifts P → E and leaves, and the A site is open for the next codon.

Trap: peptidyl transferase moves the chain from the P-site tRNA onto the amino acid on the A-site tRNA, so the lengthened chain sits in the A site until translocation; "the peptide bond forms at the P site" is an oversimplification.
Wobble: pairing is relaxed at the codon's third position (G-U pairs; inosine in an anticodon pairs with C, U or A), so one tRNA can read several codons.
Polysomes: several ribosomes translate one mRNA at once, each making its own chain.

Termination (Terminate)

A stop codon (UAA, UAG, UGA) reaches the A site. No tRNA recognizes it.
A release factor binds instead (RF-1 or RF-2 in bacteria, eRF-1 in eukaryotes) and stimulates hydrolysis of the bond between the P-site tRNA and the chain, releasing the finished polypeptide.
A nonsense mutation creates a premature stop codon and a truncated protein (see point vs frameshift vs splice-site mutations).

Drugs and toxins by the step they hit

Agent	Binds	What it blocks	Testable extra
Aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin)	30S	Fidelity: codon-anticodon mismatches give faulty proteins	Kidney toxicity (often reversible), vestibular and hearing toxicity (often irreversible), prolong the effect of neuromuscular blockers
Tetracyclines (doxycycline, minocycline; tigecycline)	30S	Aminoacyl-tRNA entry into the A site	Bacteriostatic; tooth staining and enamel hypoplasia when given during tooth development (up to age 8), although short doxycycline courses (under 21 days) are now considered acceptable at any age; photosensitivity; absorption reduced by calcium, magnesium, iron, aluminum
Linezolid	50S	Formation of the initiation complex	Myelosuppression, serotonin syndrome, peripheral and optic neuropathy
Chloramphenicol	50S	Peptide-bond formation (peptidyl transferase)	Dose-related marrow suppression, idiosyncratic aplastic anemia, gray baby syndrome
Macrolides (azithromycin, clarithromycin, erythromycin) and clindamycin	50S	Chain elongation: current texts say they block peptide-bond formation; the classic board answer is translocation	Erythromycin: GI upset, QT prolongation, CYP450 inhibition
*Diphtheria toxin, Pseudomonas* exotoxin A**	Human eEF-2	ADP-ribosylation of eEF-2, so translocation stops	A single catalytic diphtheria toxin molecule in the cytosol can kill a cell
Shiga toxin, EHEC Shiga-like toxins, ricin	Human 60S subunit (28S rRNA)	RNA N-glycosidases that remove one adenine from 28S rRNA, which disrupts elongation-factor binding and stops protein synthesis	Shiga toxins: hemorrhagic colitis and hemolytic uremic syndrome; ricin: castor beans

Coverage and toxicities by class are in the antibiotic mechanisms hub; the toxins come up again in Corynebacterium diphtheriae and the Pseudomonas question.

How USMLE tests it: answer-choice tells

"Ribosomes fail to assemble on the mRNA" or "initiator tRNA is not loaded" → initiation: eIFs, cap binding, eIF-2.
"The ribosome stalls and can't move" → translocation: EF-G (bacteria), eEF-2 (human; diphtheria toxin, exotoxin A).
"Peptidyl transferase" → large-subunit rRNA; chloramphenicol in bacteria.
"Stop codon" or "release factor" → termination; there is no stop tRNA.
"Charging" → aminoacyl-tRNA synthetase and ATP, which happens before translation.
Direction and start: mRNA read 5′ → 3′, protein made N → C; fMet in bacteria, Met in eukaryotes.

Practice question

A cell-free lysate of reticulocytes translates globin mRNA efficiently. When heme is withheld, protein synthesis soon stops. Charged tRNAs, including methionyl initiator tRNA, are present at normal levels, but 40S subunits no longer pick up the initiator tRNA and no 80S ribosomes assemble on the mRNA. Chains that had already started are completed and released normally. Which step is most directly blocked?

A. Peptidyl transferase activity of large-subunit rRNA B. eIF-2-mediated delivery of methionyl initiator tRNA to the 40S subunit C. eEF-2-mediated translocation of the ribosome D. Recognition of stop codons by eRF-1 E. Attachment of methionine to its tRNA by aminoacyl-tRNA synthetase

Answer: B. Without heme, a protein kinase that phosphorylates eIF-2 is activated, and translation stops at initiation: eIF-2-GTP normally carries Met-tRNAi to the 40S subunit, so no initiation complex and no 80S ribosome form. Ribosomes already elongating finish their chains, which is why the completed proteins are released normally.

A: peptide-bond formation is elongation, after initiation. Chains already underway were completed, so peptidyl transferase works. (Chloramphenicol blocks it in bacteria.)
C: eEF-2 drives translocation during elongation, which is also intact here. This is the eIF-2 vs eEF-2 trap: diphtheria toxin and Pseudomonas exotoxin A ADP-ribosylate eEF-2.
D: stop-codon recognition is termination, and finished chains were released normally.
E: charging happens before translation and uses ATP; charged tRNAs, including Met-tRNAi, are normal.

Key takeaways

I-E-T: Invite (initiator tRNA in the P site), Extend (A → P → E), Terminate (release factor at a stop codon).
Going APE: A = aminoacyl-tRNA arrives, P = peptidyl-tRNA, E = empty tRNA exits.
Bacteria: 70S, Shine-Dalgarno pairs with 16S rRNA, fMet, IF-2, EF-Tu, EF-G, RF-1 and RF-2. Eukaryotes: 80S, 5′ cap and scanning, Met, eIF-2, eEF-1α, eEF-2, eRF-1.
Peptidyl transferase is rRNA (a ribozyme); the new bond forms on the A-site tRNA.
ATP charges tRNA; GTP powers initiator delivery, aminoacyl-tRNA delivery and translocation.
eIF-2 = initiation; eEF-2 = translocation, the target of diphtheria toxin and Pseudomonas exotoxin A.
30S: aminoglycosides (misreading), tetracyclines (A site). 50S: chloramphenicol (peptide bond), linezolid (initiation complex), macrolides and clindamycin (translocation on boards).