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 34RNA structureThe discovery of mRNASynthesis of RNA (Transcription)• RNA polymerase• Promoters• TerminationRNA modification• Capping• Polyadenylation• Splicing• RNA degradationRNA IIThe mechanics of intron splicing - Level III=> Self-splicing introns• Catalytic RNA• Tom Cech and Sidney Altman 1981• ‘RNA world’• Other ribozymes - ribosome - spliceosomemRNA modification (PolII transcripts in eukaryotes)CappingPolyadenylation SplicingMolecular biologyImplications for gene function -> Alternative splicing yields multiple proteins from a single gene-> Splice site mutations can cause loss-of-functionImplications for evolution-> Exon shuffling can create new genesImplications of mRNA splicing - IAlternative splicing -> Multiple proteins from one geneExample: Tropomyosin5’ splice site = Splice donor site3’ splice site = Splice acceptor siteImplications of mRNA splicing – Alternative splicing Example: DystrophinGenetic defect: Duchenne Muscular Dystrophy, X-linked muscle wasting disease*A mutation in a splice-site commonly causes loss-of-function.Example: The m6 allele has a mutation in an intron-exon junction (splice site)Implications of mRNA splicing - IIImplications of mRNA splicing – IIExample:Mutant alleles in Phenylalanine hydroxylase(Phenylketonuria gene) Mutationsaffecting the proteinMutations affectingmRNA splicingAllelic SeriesIntron ExonGene 1 Gene 2Illegitimate recombinationChimeric geneThe new gene combines functions of two separate genes into one.Implications of mRNA splicing – IIINew genes can arise by illegitimate recombination between introns (exon shuffling).RNA structureThe discovery of mRNASynthesis of RNA (Transcription)RNA modification(Pol II transcripts in eukaryotes)• Capping• Polyadenylation• SplicingRNA degradationRNA IIThe fate of mRNAmRNADegradationTranslationendonucleaseexonucleases3’>5’ 5’>3’5’AAAAAAAAAAAAAAAADeadenylation\DecappingATranslational repressionA. TriggerB. SilencerC. Target7mGmiRNADicer endonucleaseRISC endonuclease siRNA (21-24 bases)RNA interference - The ABCguide RNAdouble-stranded RNAe.g. virus RNA or short hairpin RNAMello and Conte Nature 2004RNA degradation= ‘Slicer’ activitymRNATranslational repressionA. TriggerB. SilencerC. Target7mGDicer endonucleaseRNA interference - The ABCdouble-stranded RNAMello and Conte Nature 2004mRNARISC endonuclease siRNA (21-24 bases)1. RNA Viruses2. Endogenous transcripts- Bidirectional transcription- Inverted repeat genes3. Exogenous RNA- Microinjection- OtherApplicationsVirus resistant plantsExperimental toolMolecular therapy?Sources of dsRNABiological roleVirus resistance (plants)Gene regulation (development)Control of transposable elementsLecture 21October 13, 2014The Genetic CodeRefresher: ProteinsDNA -> RNA -> ProteinCodon -> Codon -> Amino acidStructure: Primary – Secondary – Tertiary - QuarternaryDNA and protein are colinear (Charles Yanofsky)Properties of the genetic code: - The genetic code is a triplet code (Francis Crick)Cracking the genetic code (Nirenberg, Ochoa, Khurana)RibosomeTranscriptionTranslationRibosomeTranslationAUG UAU AUU CAG GGU UAAMet Tyr Ile Gln Gly StopM Y I Q G mRNAPeptideAmino acid Molecule with an amino group and a carboxyl groupPeptide A few amino acids linked by amide (peptide) bondsPolypeptide = Long peptideProtein ≥ 1 polypeptide and any cofactorsDNA ATG TAT ATT CAG GGT TAATranscriptionTranslationPeptide bond formation20 types of amino acidsBond lengths in Ångstrom (1Å = 0.1nm)bond (amide)Proteins: From amino acids to functionAlpha helixBeta-sheetLoopsCo-factors• NADH• ATP• Metal ions• Chlorophyll• etcLecture 21October 13, 2014The Genetic CodeRefresher: ProteinsDNA -> RNA -> ProteinCodon -> Codon -> Amino acidStructure: Primary – Secondary – Tertiary - QuarternaryDNA and protein are colinear (Charles Yanofsky)Properties of the genetic code: - The genetic code is a triplet code (Francis Crick)Cracking the genetic code (Nirenberg, Ochoa, Khurana)Colinearity of DNA and protein (Charles Yanofsky, 1963)Case study: E. coli TrpA gene, tryptophan synthetaseGenetic mappingBiochemical analysisGiven: 15 E. coli strains with different mutant alleles of TrpA.Experiment I: Map mutations II: Isolate each mutant TrpA protein and determine the amino acid sequence1. Flow of Information DNA RNA Proteinbases (4, ACGT) Bases (4, ACGU) Amino acids (20, alanine etc...)2. Biochemical basistRNA mediates between bases and amino acids (Francis Crick’s adapter hypothesis)GENETIC CODEThe 7 habits of a highly successful genetic codeThe genetic code is …1. A triplet codeTHE POD HAD BUT ONE PEATHE,POD,HAD,BUT,ONE,PEAT HEP ODH ADB UTO NEP EA2. Commaless3. Non-overlapping4. Unambiguous5. Degenerate6. Three reading frames7. Universal (almost)THEPODHADBUTONEPEAThe Hep Epo Pod ....THE POD HAD BUT ONE PEATH EPO DHA DBU TON EPE AXXEach triplet  one amino acidOne amino acid  more than one tripletThe 7 habits of a highly successful genetic code-> Triplets 4 x 4 x 4 = 64 Example: TTTDoublet code? 4 x 4 = 16 not enough codons for 20 amino acidsQuadruplet code? 4 x 4 x 4 x 4 = 256Amino acids20 .... and a Stop-signalPhenylalanineThe triplet code: Crick and Brenner’s T4 phage experiments1. Isolate independent single-base (insertion/deletion) mutations in the rII host range gene (proflavin as mutagen). 2. Sort the mutations into (+) type and (-) type-> One (+) can suppress one (-) but 1 (+) cannot suppress 1 (+).3. !!! Three (+) type mutations can suppress each other. Double mutant generated by crossing over between single-mutant strainsThe triplet code: Crick and Brenner’s T4 phage experiments1. Isolate independent single-base Indel mutations in the rII host range gene. 2. Sort the mutations into (+) type and (-) type.-> One (+) can suppress one (-) but 1 (+) cannot suppress 1 (+).2. !!! Three (+) type mutations can suppress each other. THE POD HAD BUT ONE PEA THE POD HAD BUT ONE PEATHE POD HAA DBU TON EPE ATH EPO DHA DBU TON EPE ATHE POD HAA DBU UTO ONE PEA THE POD HAD BUT ONE PEATHE POD HAA DBU TNE PEA THE POD HAD BUT