Translation Initiation

1. The 43S Preinitiation Complex

Eukaryotic cap-dependent initiation:

1. eIF2-GTP · Met-tRNA_iforms the ternary complex; loads onto the 40S at the P site along with eIF1, eIF1A, eIF3, and eIF5. This is the 43S PIC.
2. eIF4F (cap-binding eIF4E + scaffold eIF4G + helicase eIF4A) binds the m⁷G cap and recruits the 43S PIC. PABP bound to the poly-A tail interacts with eIF4G, closing the mRNA into a “closed loop” that increases re-initiation efficiency.
3. The 43S scans 5′ → 3′ through the 5′ UTR, unwinding secondary structure with eIF4A’s ATP-dependent helicase activity.
4. At an AUG in good Kozak context(gccRccAUGG), codon-anticodon pairing is detected; eIF5 triggers eIF2 GTP hydrolysis; eIF1 is displaced; Met-tRNA_i settles into the P site.
5. eIF5B-GTP catalyses 60S joining; eIF5B hydrolyses GTP and dissociates. The 80S IC is ready for elongation.

Energy cost: 1 GTP (eIF2) + 1 GTP (eIF5B) + several ATPs (eIF4A helicase) per initiation event — far more than a single elongation cycle.

2. Regulation of eIF2 and eIF4

The two main control points:

• eIF2α phosphorylation (Ser51)by GCN2 (amino-acid starvation), PERK (ER stress), HRI (haem starvation), PKR (dsRNA/viral sensing). Phospho-eIF2α traps eIF2B as a non-productive complex, arresting most translation — the integrated stress response (ISR). Certain mRNAs with upstream ORFs (ATF4, CHOP, GCN4) are translationallyupregulated under these conditions.
• eIF4E cap availability: 4E-BPs compete with eIF4G for eIF4E binding. mTORC1-phosphorylated 4E-BPs release eIF4E, increasing cap-dependent initiation; mTORC1 inhibition (rapamycin, nutrient stress) has the opposite effect. This is how growth signalling links to ribosome use.

3. Bacterial Initiation

Bacteria use a distinct system: the Shine-Dalgarno sequence (AGGAGG) in the mRNA base-pairs with the 3′ end of 16S rRNA, positioning the start codon directly in the P site. No scanning. Three initiation factors (IF1, IF2, IF3) are used. fMet-tRNA_fMet is the initiator tRNA; the formyl group distinguishes initiator from elongator Met-tRNA and is removed by a formyl deformylase after initiation. Many bacterial mRNAs are polycistronic — multiple ORFs sharing one mRNA — with each ORF having its own SD and start codon.

4. IRES-Mediated Cap-Independent Initiation

Certain mRNAs initiate without a cap via internal ribosome entry sites (IRES): structured RNA elements that directly recruit the 40S. Four classes by eIF requirement:

• Type I (Polio, rhinovirus): requires most eIFs except eIF4E.
• Type II (EMCV, FMDV): similar, cap-independent.
• Type III (HCV, pestiviruses): only requires eIF3 + eIF2.
• Type IV (cricket paralysis virus, CrPV): requires no eIFs, no initiator tRNA — the IRES mimics Met-tRNA_i and directly recruits the 40S.

Cellular IRESs are proposed in cellular mRNAs (c-Myc, VEGF, hypoxia-response genes) that need to be translated during cap-dependent shutdown; the evidence for these is less uniform than viral IRESs.

5. uORFs and Translational Regulation

Upstream open reading frames in 5′ UTRs provide translational control: scanning ribosomes initiate at the uORF, translate it, and may or may not reinitiate at the downstream main ORF. Under normal conditions, short uORFs favour reinitiation (efficient main-ORF translation). Under stress-induced low ternary complex, the ribosome bypasses uORFs, shifting translation toward specific downstream ORFs. This is the mechanism by which yeast GCN4 and mammalian ATF4, CHOP are translationally upregulated during stress. ~50% of human mRNAs contain uORFs; they represent a broad, largely unexplored layer of post-transcriptional regulation.