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Translation

Translation . Introduction 1 -the genetic code 2 -tRNAs codon recognition and aminoacylation 3 -Translation initiation 4 -Translation elongation 5-Peptide bond formation catalysis in the ribosome 6-Translation termination 7-Quality Control of translation. Fig 30-7 Voet and Voet :

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Translation

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  1. Translation Introduction 1-the genetic code 2-tRNAs codon recognition and aminoacylation 3-Translation initiation 4-Translation elongation 5-Peptide bond formation catalysis in the ribosome 6-Translation termination 7-Quality Control of translation Fig 30-7 Voet and Voet: What’s wrong with that picture ? Not treated: Translation Regulation (initiation, elongation, termination)

  2. The Genetic Code

  3. tRNAs are adaptor molecules Crick’s Adaptor Hypothesis

  4. One tRNA can recognize several codons AntiCodonCodon (tRNA) (mRNA) C G A U U A,G G C,U I C,U,A

  5. Biological Peptide Formation is NOT a condensation reaction Biological Peptide Bond Formation Condensation

  6. Aminoacylation of tRNAs Interests: 1)Provide the adaptor for translation 2)Activates the carboxyl group of the amino acid for peptide bond formation

  7. Fidelity of tRNA Amino acylation - Discrimination of the “right” AA on a single step can not exceed 1/100 - AA tRNAsynthetases specificity occurs by two steps 1)Charging of the amino acid on the RNA in the “synthetic Domain” 2) Shuttling of the charged amino acyl-tRNA in the editing site Correct Charging This would result in errors Incorrect Charging Reynolds et al. Nat.Rev.Microbiol.2010

  8. A functional comparison between DNA Polymerase and tRNAsynthetases Synthetic mode --> Editing mode Synthetic mode --> Editing mode

  9. Prokaryotes Eukaryotes 50S 60S 2820 kDa 1590 kDa Large Subunit 28S, 5S, 5.8 S rRNAs 23S, 5S rRNAs Ribosomes Composition 49 proteins 31 proteins Peptidyl Transferase Center Peptidyl Transferase Center 30S 40S 930 kDa 1400 kDa Small subunit 16S rRNA 18 S rRNA 21 proteins 33 proteins mRNA Binding Error Correction mRNA Binding Error Correction

  10. mRNA binding Error correction 70S ribosome functions Small Subunit Large Subunit Peptidyl transferase Cates et al., Science (1999)

  11. Prokaryotic translation initiation Translation immediately follows transcription in Prokaryotes Translation of a polycistronic mRNA in prokaryotes ORF1 ORF2 ORF3 Stop AUG SD Translation stops Translation starts Ribosome Dissociates Ribosome Binds to Shine-Dalgarno sequence

  12. Translation immediately follows transcription in Prokaryotes From the relative sizes of the nascent polypeptides, can you pinpoint the direction of translation and the 5’-3’ polarity of the mRNA on this electron micrograph ?

  13. Eukaryotic translation initiation 1) Binding of the mRNA by the 40S subunit+initiation factors 2) Scanning of the mRNA to search for AUG 3) Binding of the 60S subunit

  14. Recognition of the 5’-cap structure of eukaryotic mRNAs by eIF4e Cap binding pocket is on the concave surface of the protein – allows easy entry of the mRNA 5’-end PDB ID = 1L8B Niedzwiecka et al. J.Mol.Biol. 2002 • base sandwich-stacking between Trp56 and Trp102 • formation of three Watson–Crick-like hydrogen bonds with Glu103 and backbone NH of Trp102 = H20 • van der Waals contact of the N(7)-methyl group with Trp166

  15. Simplified view of the elongation and termination reactions in the ribosome Leung et al. Ann.Rev. Biochem. 2010

  16. Ribosome Recycling Factor (RRF) resembles a tRNA molecule A case of molecular mimickry ! This allows RRF to enter the empty A site dyring translation termination Blue = RRF Red = yeast tRNAPhe PDB ID = 1DD5 Selmer et al. Science 1999:Vol. 286. no. 5448, pp. 2349 - 2352

  17. Translation Elongation in Prokaryotes: 2 independentFidelity Mechanisms Normal Elongation(codons and tRNAs are matched –see color code) Aberrant Elongation Incorporation of an incorrect amino acids promotes an even greater loss of fidelity for the next round of elongation, and ultimately triggers peptide release and dissociation Reynolds et al. Nat.Rev.Microbiol.2010

  18. Peptide Bond Formation: The Peptidyl Transferase Reaction

  19. Evidences for RNA Catalysis in the Peptidyltransferase center Biochemical Evidence: SubunitCatalytic Efficiency 50S E.coli subunit 100% Deproteinized 50S E.coli 20% 50S T.aquaticus 100% Deproteinized 50S T.aquaticus 80% Deproteinized 50S T.aquaticus+RNAse 0% Deproteinized = 95% of the proteins are removed Structural Evidence: •There is no protein with >10 angstrom of the catalytic center of the peptidyltransferase reaction in the 50S subunit ---> The reaction has to be catalyzed by RNA ---> 1 Adenine base is perfectly localized for this. Yellow = Proteins Grey = RNA Green = Peptidyl Transferase Center

  20. Proposed Catalysis Model in the Peptidyltransferase center T.Steitz - Nature Reviews Mol.Cell.Biol. Vol.9 - March 2008 Proposed Catalysis Model : • crucial role of the 2’hydroxyl of A76 of the tRNAas a proton Shuttle” in receiving a proton from the  amino group (A site - labeled “N”) and transferring it to the 3’O of the tRNA-peptide link in the P site Leung et al. Ann.Rev. Biochem. 2010

  21. Quality Control of Translation: These quality control pathways prevent the accumulation of truncated or deficient proteins due to defects in mRNAs: - truncated mRNAs (because of random breaks in RNAs) - mRNAs that do not contain stop codons (because of mistakes in the 3’-end processing reactions) - mRNAs containing premature termination codons (gene mutations or errors made by RNA polymerase) 2 examples of Translation QC in eukaryotes • “Nonstop” protein degradation in eukaryotes •  Nonsense mediated mRNA decay in eukaryotes

  22. Qualitycontrol of translationfor “non-stop” mRNAs • Non Stop mRNAs can be generated if the poly(A) tail is added before the stop codons (mistakes by the C&P machinery) Proteasome • The Poly(A) tail is translated as a Poly-Lysine tract • Poly-Lysine tract sticks in the ribosome exit tunnel because of electrostatic interactions • Ltn1 ubiquitinligase attaches polyubiquitin chains to the nascent polypeptide stuck to the ribosome • This marks the aberrant protein for degradation by the proteasome Bengston & Joazeiro – Nature 2010 - doi: 10.1038/nature09371

  23. Premature termination codons (PTC) are frequently associated with genetic diseases • >90% of mutations associated with: • Duchenne muscular dystrophy • Familial adenomatouspolyposis • Hereditary desmoid disease • Ataxia telangiectasia • Hereditary breast and ovarian cancers • Polycystic kidney disease • >75% of mutations associated with: • Emery-Dreifuss muscular dystrophy • Fanconi anemia • Non-polyposis colorectalcancer No detection of the truncated proteins The corresponding mRNA is unstable and degraded by a mechanism called Nonsense Mediated mRNA Decay(NMD) coupled to translation

  24. Simplified view of the mechanism of Nonsense Mediated Decay (NMD) in eukaryotic cells Translation and Termination proceed normally STOP AUG m7G (A)n “Normal” mRNA Recruitment of RNA degradative enzymes that prevent accumulation of PTC-containing mRNAs Assembly of a complex that signals NMD Upf3 Upf2 Upf1 STOP AUG STOP m7G (A)n Ribosome Stalled at Premature Termination Codon Aberrant mRNA containing premature stop codon (PTC) mRNA Destruction

  25. Translation and 153B have been terminated !

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