PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Start codonOpen Reading frame (ORF) = Coding sequence (CDS)Stop codonUAA, UAG or UGAAUG ccc cgg uau gca ggg uac gug aaa ..... UAAAUGuaugcagggguccaaugg..aaaanCapOrganization of a typical protein-coding mRNACodons/Triplets frame 3 frame 2What is a frameshift mutation?I. Homopolymers RNA composition Protein compositionPoly-U (UUUUUU....) Phenylalanine (100%)Poly-A Lysine (100%)Poly-C Proline (100%)The cracking of the genetic code Severo Ochoa, Marshall Nirenberg, and Gobind Khorana, 1960’sPrinciple:1. Synthesize RNA of defined basepair composition2. Add RNA to translation-competent E. coli cell extract (in vitro assay)3. Determine which amino acids are incorporated, and in which proportion.4. Deduce which codons stand for which amino acids.UUU PhenylalanineAAA LysineCCC ProlinePolynucleotide phosphorylaseRNA + phosphate NDPsReaction is reversible!Synthesize RNA from nucleoside-diphosphate monomers100% UDP -> UUUUUUUUUUUUUUU... 80% GDP 20% CDP -> GGGCGGGGCGGCGGGCGGGG...2. Mixed co-polymers = Random polymers from ‘spiked’ mixtures (e.g. 90%U, 10%G).UUU 73 % PheUUG, UGU, GUU 8.1% each of Leu, Cys, Val, respectivelyUGG, GUG, GGU 0.9% each of Trp, Val, Gly, respectivelyGGG 0.1% GlyThe cracking of the genetic code (continued)3. Defined polymers Example: Poly (GU)  GUGUGUGUGUGUGUGUGUGGUG and UGU 50% each of Val and Cys Conclusion: UGU=Cysteine; GUG = ValineConclusion: GGG=GlycineThe cracking of the genetic code (continued)3. Defined polymers Example: Poly (GAUA) GAU, AUA, UAG, AGA no incorporation of amino acids !Example: 5’ -AUG + tRNA~Met + Ribosome  Ribosome 5’AUG  tRNA~Met(Met radioactive) --------------- Complex ---------------- 5’ -AUG + tRNA~Phe + Ribosome  No complex Radioactive Phe is washed through a filterRadioactive Met is retained on a filter4. Single triplet binding assay Conclusion: one the 4 codons must be a ‘nonsense’ codon= stop signal!Conclusion: AUG codes for Methionine, and not PhenylalanineThe cracking of the genetic code (continued)Example: 5’ -AUG + tRNA~Met + Ribosome  Ribosome 5’AUG  tRNA~Met(Met radioactive) --------------- Complex ---------------- 5’ -AUG + tRNA~Phe + Ribosome  No complex Radioactive Phe is washed through a filterRadioactive Met is retained on a filter4. Single triplet binding assay Conclusion: AUG codes for Methionine, and not PhenylalanineAUGAUGUACUACMet~metStart codonOpen Reading frame (ORF) = Coding sequence (CDS)Stop codonUAA, UAG or UGAAUG ccc cgg uau gca ggg uac gug aaa ..... UAAAUGuaugcagggguccaaugg..aaaanCapOrganization of a typical protein-coding mRNACodons/Triplets frame 3 frame 2What is a frameshift mutation?The genetic code is ‘non-overlapping’? Why might it be reasonable to suspect that the code is overlapping?? How to prove that the code is non-overlapping?Ile Leu CysIle Ala GlnTranslation = Protein synthesis• Structure of proteins• Properties of the genetic code• Cracking the genetic code• Ingredients for protein synthesis tRNA The ribosome• Translation in bacteria (prokaryotes) Initiation Elongation Termination• Translation in eukaryotesTranslation - The prokaryotic (70S) ribosomeGFScodonsanticodonsmRNAtRNAIngredients for translation - I : tRNA**2D: Cloverleaf3D: LIngredients for translation - I B: Aminoacyl-tRNA-SynthetasesAmino acidtRNA AlaATP AMP + PPiAla~tRNAAlaAlanine +Charged tRNAAlanineAminoacyl-tRNA- SynthetaseThere are 20 different Aminoacyl-tRNA Synthetases ribose 3’ C – O – C – C – NH3+OCH3---How many different tRNAs does the cell need?How many different tRNAs does the cell need?GTranslation = Protein synthesis• Structure of proteins• Properties of the genetic code• Cracking the genetic code• Ingredients for protein synthesis tRNA The ribosome• Translation in bacteria (prokaryotes) Initiation Elongation Termination• Translation in eukaryotesIngredients for translation - II : Ribosomesetc.Ingredients for translation - II : Ribosomal RNArRNA secondary structureExtended basepaired regionsLoopsBubblesExample:16S rRNAbasepaired/helical regionsbubbleloopIngredients for translation - II : Large (50S) Ribosomal SubunitView of thesubunit interface side- tRNA- anticodon loop- peptide and amino acidrRNAProteinsX-raycrystalstructureIngredients for translation - II : The prokaryotic (70S) ribosomePeptide exit channelA = Aminoacyl siteP = Peptidyl siteE = Exit siteCryo-electron microscopyTranslation - The prokaryotic (70S) ribosomeTranslation initiation: Prokaryotes (Overview)Translation initiation in prokaryotes: The Shine-Dalgarno sequence brings together the mRNA and the 30S ribosome.It positions the ribosome such that the P-site overlaps the AUG start codon. tRNAfMet Note: No SD sequence in eukaryotesTranslation initiation in prokaryotes: The 30S ribosome harbors a tRNAcharged with methionine.Initiation factors are neededfor 50S subunit joining.Translation initiation in Eukaryotes : Ribosome scanningInitiation factor 2 (eIF2~GTP) delivers the methionyl-tRNA to the 40S.The 40S binds the 5’cap and ‘scans’ the mRNA for the first AUG codon.ATP hydrolysis may be needed tomelt RNA secondary structure.Once the AUG has been recognized (eIF2-GDP+phosphate release) ...... the 60S subunit joins. An initiation factor (eIF5B~GTP) is needed for this.The 80S ribosome (P-site) sits on the AUGstart codon. Why does this process move ‘forward’ inone direction? “Brownian ratchet”Translation elongation (Prokaryotes)GTP-hydrolysisGTP- hydrolysisEF-G leavestRNA in theE(xit) site leaves with EF-Tu4 StepsTranslation elongation: The ribosome is a ribozymePeptidyl~tRNAPP-site+ Ala~tRNAAlaA-sitePeptidyl~tRNAAlaA-site+ tRNAPRibosomeThe active site of the ribosome:= Peptidyltransferase centerLarge subunit (50S or 60S)Ribosomal RNA in active site!No proteinNo ATPP-site+H2OTranslation elongation (Prokaryotes) - Structural models*Antibiotics that inhibit translationKasugamycin Inhibits binding of initiator tRNA to 30SErythromycin Binds 50S and prevents it from joining the