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Eukaryotic Translation Initiation: Mechanisms and Factors

The process of translation initiation in eukaryotes involves a complex interplay of molecular components, including the 40S and 60S ribosomal subunits, mRNA with a distinctive 5' cap and 3' polyA tail, and various initiation factors (eIFs). Key elements such as the Kozak sequence and the initiator tRNA (Met-tRNAiMet) are essential for the recognition of the start codon (AUG). This article explores the roles of proteins like eIF4E, eIF4G, and eIF3, as well as the impact of phosphorylation on translation initiation regulation. It also addresses alternative initiation mechanisms, particularly Internal Ribosome Entry Sites (IRES).

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Eukaryotic Translation Initiation: Mechanisms and Factors

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  1. VALGUSÜNTEESI INITSIATSIOON EUKARÜOOTIDES

  2. INITSIATSIOON • 40S subunit • mRNA • Initsiator tRNA Met-tRNAiMet • 10 eukaryotic initsiation factors • PolyA-binding protein • 60S subunit

  3. 40S subunit • 18S rRNA • No anti-Shine-Dalgarno sequence • DOES NOT RECOGNIZE mRNA START SITE

  4. mRNA • Monocystronic • CAP structure at the 5’ terminus • AUG start codon (Kozak sequence) • PolyA sequence at the 3’ terminus A/GNNAUG ORF CAP AAUAAA AAAAAAAAA 5’ 3’

  5. mRNA CAP • Promotes recognition of the translation initiation site by the 40S subunit • Recognized by eIF4E eIF4E CAP

  6. mRNA CAP • Capping is linked to the early stages of transcription initiation and elongation. • A phosphohydrolase removes the gamma phosphate from the 5' end of the transcribed pre-mRNA. • Guanylyl transferase catalyzes the condensation of GTP with the 5' end of the pre-mRNA. This creates the unusual 5'-5' triphosphate linkage. Pyrophosphate is released. • The terminal guanosine nucleotide is methylated by guanine-7-methyl transferase. S-adenosyl-methionine is required as a cofactor for this reaction.

  7. mRNA Kozak sequence • A/GNNAUGG • Promotes selection of right AUG start codon • Situated 40-100nt away from the CAP

  8. mRNA PolyA sequence • 150-200nt • Binds polyA-binding protein (PABP) that promotes mRNA 5’ – 3’ ends interaction that stimulate initiation of translation IF4G PABP IF4E CAP polyA

  9. Initsiator tRNA Met-tRNAiMet • Met-tRNAiMet • Not formylated • A1 : U72 base pair is important for right initiator tRNA selection • Three conserved G:C base pairs in the anticodon stem

  10. eIF4A+eIF4G +eIF4E=eIF4F

  11. 43S

  12. 48S

  13. INITIATION FACTORS eIF4F • attaches 40S to mRNA eIF4G eIF4A eIF4E • 80S formation • IF release eIF5B • adapter • binds PABP • binds 43S • binds CAP • Helicase bind 40S eIF3 eIF5 eIF1 eIF1A eIF2 • holds subunits • apart • attaches 40S to mRNA • Met-tRNAi • scanning • AUG selection • 40S recruitment • adapter • binds eIF3, eIF1 and eIF2 • induces GTP hydrolysis

  14. IRES (Internal Ribosome Entry Site) • 40S subunits bind mRNA in a CAP independent manner • Does not involve scanning

  15. IRES (Internal Ribosome Entry Site) • Initiation factor dependent internal initiation • eIF3, eIF2, Met-tRNAi dependent internal initiation • Initiation factor and Met-tRNAi independent internal initiation

  16. IRESInitiation Factor Dependent Internal Initiation • Encephalomyocarditis virus; Poliovirus; FGF2; eIF4G

  17. IRES eIF3, eIF2, Met-tRNAiDependent Internal Initiation • Hepatitis C virus, GB virus

  18. IRES (Initiation factor and Met-tRNAi independentEntry Site) • Dicistroviruses: Cricet paralysis virus, Taura shrimp virus • 80S ribosomes can initiate translation

  19. TRANSLATION INITIATION CONTROLL: eIF2 phosphorylation • Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of eIF2B • eIF2 is always present in excess of eIF2B

  20. TRANSLATION INITIATION CONTROLL: eIF2 phosphorylation

  21. TRANSLATION INITIATION CONTROLL: eIF2 phosphorylation • GCN4 – transcriptional activator of amino acid biosynthetic genes • GCN2 production is increased under amino acid starvation conditions • which activate GCN2

  22. TRANSLATION INITIATION CONTROLL: eIF4E inhibitory proteins

  23. eIF4E inhibitory proteins: 4E-BP • Compete with eIF4G for a common binding site on eIF4E • Binding is regulated by phosphorylation : • hypophosphorylation – prevents eIF4G binding to eIF4E (growth factors, mitogens) • hyperphosphorylation – 4E-BP does not bind eIF4E (nutrient deprivation, stress)

  24. eIF4E inhibitory proteins: MASKIN • 3’ end of most mRNA terminate with 150-200 nt. polyA tail • In frog oocytes arrested at the end of meiotic prophase polyA tail is only 20-40 nt • When the oocytes are stimulated to re-enter the meiotic divisions the polyA tails are elongated to about 150 bases

  25. eIF4E inhibitory proteins: MASKIN • Cytoplasmic polyadenylation • is controlled by CPE (cytoplasmic • polyadenylation element) • CPEB binds CPE • CPEB binds MASKIN • MASKIN binds eIF4E and prevents • eIF4G from binding eIF4G • When oocytes are induced to • complete meiosis, Aurora A catalyses • CPEB phosphorylation • CPEB stimulates polyA tail growth • Newly elongated polyA binds PABP • PABP interacts with eIF4E and • displaces MASKIN • eIF4G binds

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