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VALGUSÜNTEESI INITSIATSIOON EUKARÜOOTIDES

VALGUSÜNTEESI INITSIATSIOON EUKARÜOOTIDES. INITSIATSIOON. 40S subunit mRNA Initsiator tRNA Met-tRNA i Met 10 eukaryotic initsiation factors PolyA-binding protein 60S subunit. 40S subunit. 18S rRNA No anti-Shine-Dalgarno sequence DOES NOT RECOGNIZE mRNA START SITE. mRNA.

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VALGUSÜNTEESI INITSIATSIOON EUKARÜOOTIDES

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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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