Central dogma of molecular biology

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Central dogma of molecular biology

  1. 1. Chapter 12: From DNA to Protein: Genotype to PhenotypeCentral Dogmain Molecular Biology
  2. 2. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA and Its Role in HeredityDNA to Protein:Genotype to Phenotype
  3. 3. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe central dogmaDNA structureDNA replicationRNA structureRNA synthesis (Transcription)The genetic codeProtein synthesis (Translation)MutationConsequences of mutationLecture 1Lecture 2Lecture 3Lecture 4
  4. 4. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe Central Dogma The Flow of Information: DNA → RNA →proteinDNA ReplicationTranscription Translation A gene is expressed in two steps: DNA is transcribed to RNA Then RNA is translated into protein.
  5. 5. DNA ReplicationDNA Replication
  6. 6. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNADNA Discovery of the DNA double helixDNA double helixA. 1950’sB. Rosalind Franklin - X-ray photo of DNA.C. Watson and Crick - described the DNAmolecule from Franklin’s X-ray.
  7. 7. Chapter 12: From DNA to Protein: Genotype to PhenotypeQuestion:Question: What isWhat is DNADNA??
  8. 8. Chapter 12: From DNA to Protein: Genotype to PhenotypeDeoxyribonucleic AcidDeoxyribonucleic Acid (DNA)(DNA) Made up of nucleotidesnucleotides (DNA molecule) in a DNADNAdouble helix.double helix. NucleotideNucleotide::1. Phosphate groupPhosphate group2. 5-carbon sugar5-carbon sugar3. Nitrogenous baseNitrogenous base ~2 nm wide~2 nm wide
  9. 9. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA NucleotideDNA NucleotideOO=P-OOPhosphatePhosphateGroupGroupNNitrogenous baseNitrogenous base(A, G, C, or T)(A, G, C, or T)CH2OC1C4C3C25SugarSugar(deoxyribose)(deoxyribose)
  10. 10. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA Double HelixDNA Double HelixNitrogenousNitrogenousBase (A,T,G or C)Base (A,T,G or C)““Rungs of ladder”Rungs of ladder”““Legs of ladder”Legs of ladder”Phosphate &Phosphate &Sugar BackboneSugar Backbone
  11. 11. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA Double HelixDNA Double HelixPPPOOO123455335PPPOOO12 3455353G CT A
  12. 12. Chapter 12: From DNA to Protein: Genotype to PhenotypeNitrogenous BasesNitrogenous Bases PURINESPURINES1. Adenine (A)Adenine (A)2. Guanine (G)Guanine (G) PYRIMIDINESPYRIMIDINES3. Thymine (T)Thymine (T)4. Cytosine (C)Cytosine (C) T or CA or G
  13. 13. Chapter 12: From DNA to Protein: Genotype to PhenotypeBASE-PAIRINGSBASE-PAIRINGSBase # ofPurines Pyrimidines Pairs H-BondsAdenine (A)Adenine (A) Thymine (T)Thymine (T) A = T 2Guanine (G)Guanine (G) Cytosine (C)Cytosine (C) C G 3CG3 H-bonds
  14. 14. Chapter 12: From DNA to Protein: Genotype to PhenotypeBASE-PAIRINGSBASE-PAIRINGSCGH-bondsT A
  15. 15. Chapter 12: From DNA to Protein: Genotype to PhenotypeChargaff’s RuleChargaff’s Rule AdenineAdenine must pair with ThymineThymine GuanineGuanine must pair with CytosineCytosine Their amounts in a given DNA molecule will beabout the sameabout the same.G CT A
  16. 16. Chapter 12: From DNA to Protein: Genotype to PhenotypeQuestion:Question: If there is 30% AdenineAdenine, how muchCytosineCytosine is present?
  17. 17. Chapter 12: From DNA to Protein: Genotype to PhenotypeAnswer:Answer: There would be 20% CytosineCytosine.Adenine (30%)Adenine (30%) == Thymine (30%)Thymine (30%)Guanine (20%)Guanine (20%) == Cytosine (20%)Cytosine (20%)(50%) = (50%)(50%) = (50%)
  18. 18. Chapter 12: From DNA to Protein: Genotype to PhenotypeQuestion:Question: When and where doesWhen and where does DNA ReplicationDNA Replicationtake place?take place?
  19. 19. Chapter 12: From DNA to Protein: Genotype to PhenotypeSynthesis Phase (S phase)Synthesis Phase (S phase) S phase in interphase of the cell cycle. Nucleus of eukaryotesMitosis-prophase-metaphase-anaphase-telophaseG1 G2SphaseinterphaseDNA replication takesDNA replication takesplace in the S phase.place in the S phase.
  20. 20. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Origins of replicationOrigins of replication1. Replication ForksReplication Forks: hundredshundreds of Y-shapedY-shapedregions of replicating DNA moleculesreplicating DNA moleculeswhere new strands are growing.ReplicationReplicationForkForkParental DNA MoleculeParental DNA Molecule3’5’3’5’
  21. 21. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Origins of replicationOrigins of replication2. Replication BubblesReplication Bubbles:a. HundredsHundreds of replicating bubbles(Eukaryotes)(Eukaryotes).b. SingleSingle replication fork (bacteria).(bacteria).Bubbles Bubbles
  22. 22. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Strand SeparationStrand Separation:1.1. HelicaseHelicase: enzyme which catalyze theunwindingunwinding and separationseparation (breaking H-Bonds) of the parental double helix.2.2. Single-Strand Binding ProteinsSingle-Strand Binding Proteins: proteinswhich attach and help keep the separatedstrands apart.
  23. 23. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Strand SeparationStrand Separation:3.3. TopoisomeraseTopoisomerase: enzyme which relievesrelievesstressstress on the DNA moleculeDNA molecule by allowing freerotation around a single strand.EnzymeDNAEnzyme
  24. 24. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Priming:Priming:1.1. RNA primersRNA primers: before new DNA strands canform, there must be small pre-existingprimers (RNA)primers (RNA) present to start the addition ofnew nucleotides (DNA Polymerase)(DNA Polymerase).2.2. PrimasePrimase: enzyme that polymerizes(synthesizes) the RNA Primer.
  25. 25. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:1.1. DNA PolymeraseDNA Polymerase: with a RNA primerRNA primer in place,DNA Polymerase (enzyme) catalyze thesynthesis of a new DNA strand in the 5’synthesis of a new DNA strand in the 5’ to 3’to 3’directiondirection.RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotideNucleotide5’5’ 3’
  26. 26. Chapter 12: From DNA to Protein: Genotype to PhenotypeRemember!!!!Remember!!!!OO=P-OOPhosphatePhosphateGroupGroupNNitrogenous baseNitrogenous base(A, G, C, or T)(A, G, C, or T)CH2OC1C4C3C25SugarSugar(deoxyribose)(deoxyribose)
  27. 27. Chapter 12: From DNA to Protein: Genotype to PhenotypeRemember!!!!!Remember!!!!!PPPOOO123455335PPPOOO12 3455353G CT A
  28. 28. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:2.2. Leading StrandLeading Strand: synthesized as asingle polymersingle polymer in the 5’ to 3’ direction5’ to 3’ direction.RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides3’5’5’
  29. 29. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:3.3. Lagging StrandLagging Strand: also synthesized inthe 5’ to 3’ direction5’ to 3’ direction, but discontinuouslydiscontinuouslyagainst overall direction of replication.RNA PrimerRNA PrimerLeading StrandLeading StrandDNA PolymeraseDNA Polymerase5’5’3’3’Lagging StrandLagging Strand5’5’3’3’
  30. 30. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:4.4. Okazaki FragmentsOkazaki Fragments: series of shortsegments on the lagging strand.lagging strand.Lagging StrandRNARNAPrimerPrimerDNADNAPolymerasePolymerase3’3’5’5’Okazaki FragmentOkazaki Fragment
  31. 31. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:5.5. DNA ligaseDNA ligase: a linking enzyme thatcatalyzes the formation of a covalent bondfrom the 3’ to 5’ end3’ to 5’ end of joining stands.Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.Lagging StrandOkazaki Fragment 2Okazaki Fragment 2DNA ligaseDNA ligaseOkazaki Fragment 1Okazaki Fragment 15’5’3’3’
  32. 32. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:6.6. ProofreadingProofreading: initial base-pairing errors areusually corrected by DNA polymeraseDNA polymerase.
  33. 33. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA ReplicationDNA Replication Semiconservative Model:Semiconservative Model:1. Watson and Crick showed:Watson and Crick showed: the two strands of theparental molecule separate, and each functions asa template for synthesis of a new complementarystrand.Parental DNADNA TemplateNew DNA
  34. 34. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA RepairDNA Repair Excision repair:Excision repair:1. Damaged segment is excisedexcised by a repairrepairenzymeenzyme (there are over 50 repair enzymes).2. DNA polymeraseDNA polymerase and DNA ligaseDNA ligase replace andbond the new nucleotides together.
  35. 35. Chapter 12: From DNA to Protein: Genotype to PhenotypeQuestion: What would be the complementary DNAstrand for the following DNA sequence?DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’
  36. 36. Chapter 12: From DNA to Protein: Genotype to PhenotypeAnswer:Answer:DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’DNA 3’-CGCATAC-5’DNA 3’-CGCATAC-5’
  37. 37. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe central dogmaDNA structureDNA replicationRNA structureRNA synthesis (Transcription)The genetic codeProtein synthesis (Translation)MutationConsequences of mutationLecture 1Lecture 2Lecture 3Lecture 4TOPICS
  38. 38. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA and RNA differ RNA differs from DNA in three ways: RNA is single-stranded (but it can fold backupon itself to form secondary structure, e.g.tRNA) In RNA, the sugar molecule is ribose ratherthan deoxyribose In RNA, the fourth base is uracil rather thanthymine.
  39. 39. Chapter 12: From DNA to Protein: Genotype to PhenotypeDNA RNA1OHOHOHOH2UH3
  40. 40. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe Central Dogma The Flow of Information: DNA → RNA →proteinDNA ReplicationTranscription Translation RNA is synthesized via a process calledTranscription mRNA, rRNA and tRNA are transcribed bysimilar mechanismsTranscription
  41. 41. Chapter 12: From DNA to Protein: Genotype to PhenotypeThree types of RNA are involved inprotein synthesisMessenger RNA[mRNA]- the templateRibosomal RNA [rRNA]- structural component ofthe ribosomeTransfer RNA [tRNA]- the adapter
  42. 42. Chapter 12: From DNA to Protein: Genotype to Phenotype
  43. 43. Chapter 12: From DNA to Protein: Genotype to PhenotypeFigure 12.7Transfer RNA - the adapterRNA is single-stranded but it can fold backupon itself to form secondary structures.
  44. 44. Chapter 12: From DNA to Protein: Genotype to Phenotype Transcription has three phases: Initiation Elongation Termination RNA is transcribed from a DNA templateafter the bases of DNA are exposed byunwinding of the double helix. In a given region of DNA, only one of thetwo strands can act as a template fortranscription.Transcription: DNA-Directed RNASynthesis
  45. 45. Chapter 12: From DNA to Protein: Genotype to PhenotypeFigure 12.4 – Part 1
  46. 46. Chapter 12: From DNA to Protein: Genotype to Phenotype Three phases: Initiation, Elongation,Termination Unwind the DNA template: template andcomplementary strands Initiation: RNA polymerase recognizes andbinds to a promoter sequence on DNATranscription: DNA-Directed RNASynthesis - Initiation
  47. 47. Chapter 12: From DNA to Protein: Genotype to PhenotypeFigure 12.4 – Part 1
  48. 48. Chapter 12: From DNA to Protein: Genotype to Phenotype Initiation Elongation: RNA polymerase elongates thenascent RNA molecule in a 5’-to-3’ direction,antiparallel to the template DNA• Nucleotides are added by complementarybase pairing with the template strand• The substrates, ribonucleoside triphosphates,are hydrolyzed as added, releasing energy forRNA synthesis.Transcription: DNA-Directed RNASynthesis - Elongation
  49. 49. Chapter 12: From DNA to Protein: Genotype to PhenotypeFigure 12.4 – Part 1
  50. 50. Chapter 12: From DNA to Protein: Genotype to Phenotype(DNA Replication figure adapted for Transcription )OHOHOHOHOHOHOHOHOHOHRNA RNADNAU U
  51. 51. Chapter 12: From DNA to Protein: Genotype to Phenotype Initiation Elongation Termination: Special DNA sequences andprotein helpers terminate transcription. The transcript is released from the DNA. This Primary Transcript is called the “pre-mRNA” The pre-mRNA is processed to generate themature mRNATranscription: DNA-Directed RNASynthesis - Termination
  52. 52. Chapter 12: From DNA to Protein: Genotype to PhenotypeFigure 12.4 – Part 2
  53. 53. Chapter 12: From DNA to Protein: Genotype to Phenotype The central dogma DNA structure DNA replication RNA structure RNA synthesis (Transcription) The genetic code Protein synthesis (Translation) Mutation Consequences of mutationLecture 1Lecture 2Lecture 3Lecture 4Topics
  54. 54. Chapter 12: From DNA to Protein: Genotype to PhenotypeTranslation
  55. 55. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe Central Dogma The Flow of Information: DNA → RNA →proteinDNA ReplicationTranscription Translation A gene is expressed in two steps: DNA is transcribed to RNA Then RNA is translated into protein.
  56. 56. Chapter 12: From DNA to Protein: Genotype to PhenotypeTranslation- the synthesis of protein from an RNAtemplate.Five stages:Pre-initiationInitiationElongationTerminationPost-translational modificationComplicated: In eukaryotes, ~300 molecules involvedTranslation
  57. 57. Chapter 12: From DNA to Protein: Genotype to PhenotypemRNA- serves as a template codetRNA- serves as an adapter moleculerRNA- holds molecules in the correctposition, protein portion also catalyzereactionsFunctions of the Types of RNA
  58. 58. Chapter 12: From DNA to Protein: Genotype to PhenotypeShine-Dalgarno sequence~10 nt upstream of initiation codonPositions ribosome at correct start sitemRNA Structure
  59. 59. Chapter 12: From DNA to Protein: Genotype to PhenotypeAll tRNA molecules have a similar but not identicalstructure- “cloverleaf”Acceptor arm- CCA-3’an amino acid will be esterified to 3’ OH of ATΨC arm - named for ribothymidine-pseudouridine-cytidine sequenceExtra arm - variable in size ~3-~20 nttRNA Structure
  60. 60. Chapter 12: From DNA to Protein: Genotype to Phenotypeanti-codon armnamed for 3 bases which base-pair withmRNA codonD arm- dihydro-uridine base modificationSequence differs for the different amino acid-not just in the anticodon armtRNA Structure, cont’d
  61. 61. Chapter 12: From DNA to Protein: Genotype to PhenotypeTriplet codonsUniversal (almost)CommalessDegenerate- wobbleUnambiguousReading framesEmbedded genesThe Genetic Code
  62. 62. Chapter 12: From DNA to Protein: Genotype to PhenotypePre-initiation - Charging thetRNA
  63. 63. Chapter 12: From DNA to Protein: Genotype to PhenotypeAminoacyl-tRNA Synthetase One for each amino acid 2 step mechanism attach a.a. to AMP transesterify to 3’ (or 2’ and then rearrange) Proofread identity elements “sieve” Modify Met-tRNAfmetto fMet-tRNAfmet
  64. 64. Chapter 12: From DNA to Protein: Genotype to PhenotypePre-initiation1. Charging the tRNA2. Formylation of met-tRNAfmet
  65. 65. Chapter 12: From DNA to Protein: Genotype to PhenotypePre-initiation1. Charging the tRNA2. Formylation of met-tRNAfmet3. Dissociation of ribosomes (IF-1 and IF-3)4. IF-2:GTP binary complex formation5. IF-2:GTP:charged tRNA ternary complexformation6. IF4F, 4A and 4B bind mRNA to place it onsmall subunit7. 40S initiation complex
  66. 66. Chapter 12: From DNA to Protein: Genotype to PhenotypeInitiationPreinitiation complexes form an 80Scomplex:small subunit, ternary complex (GDP + Pileave), mRNA, large subunit, aminoacyltRNAP-site- only thing that can enter is a peptideIn prokaryotes, f-met “tricks” the ribosomeA-site- only thing that can enter is anaminoacyl tRNA
  67. 67. Chapter 12: From DNA to Protein: Genotype to PhenotypeEach ribosome contains 3 binding sites for tRNAmolecules:A-site = aminoacyl-tRNAP-site = peptidyl-tRNAE-site = exit
  68. 68. Chapter 12: From DNA to Protein: Genotype to Phenotype07_32_initiation.jpg
  69. 69. Chapter 12: From DNA to Protein: Genotype to PhenotypeRibosome composed of 2 subunits:Small subunit – matches the tRNAs to the codonsof the mRNALarge subunit – catalyzes the formation of thepeptide bonds between aminoacids in the growing polypeptidechainThe two subunits come together near the 5’ endof the mRNA to begin synthesis of a proteinThen ribosome moves along, translating codons,until 2 subunits separate after finishing
  70. 70. Chapter 12: From DNA to Protein: Genotype to Phenotype07_28_ribosome.jpg
  71. 71. Chapter 12: From DNA to Protein: Genotype to Phenotype07_29_binding.site.jpg
  72. 72. Chapter 12: From DNA to Protein: Genotype to PhenotypeElongation1. EF-1:GTP: aminoacyl- tRNA ternarycomplex enters A-site; GDP + Pi leave(EF-Tu and EF-Ts involved with GTPmetabolism in prokaryotes)2. Peptide bond forms as P-site content istransferred onto A-site occupant3. Translocation requires GTP; GDP + Pi areproducts
  73. 73. Chapter 12: From DNA to Protein: Genotype to Phenotype07_34_stop codon.jpg
  74. 74. Chapter 12: From DNA to Protein: Genotype to Phenotype07_30_3_step_cycle.jpgPeptidyl transferasecatalyzes peptidebond formation
  75. 75. Chapter 12: From DNA to Protein: Genotype to Phenotype07_35_polyribosome.jpgA polyribosome from aeucaryotic cell
  76. 76. Chapter 12: From DNA to Protein: Genotype to PhenotypeTermination1. UAA, UAG, UGA is enveloped by A-site ofribosome2. RF-1 enters A site3. GTP is hydrolyzed, H2O is used to cleaveprotein off tRNA4. Components are recycled to synthesizeanother protein molecule
  77. 77. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe ribosome is a ribozymeDetermination of its 3-D structure in 2000 showedthat the rRNAs are responsible for:-- ribosome’s overall structure-- its ability to position tRNAs on the mRNA-- its catalytic function in forming peptide bonds(via a highly structured pocket that preciselyorients the elongating peptide and the chargedtRNA)RNA rather than protein served as first catalysts,and ribosome is a relic of an earlier time
  78. 78. Chapter 12: From DNA to Protein: Genotype to Phenotype07_31_ribos_shape.jpg
  79. 79. Chapter 12: From DNA to Protein: Genotype to PhenotypeCodons in mRNA signal where to start and stopprotein synthesisTranslation begins with codon AUG and a specialtRNA required for initiation—The initiator tRNA always carries methionine(Met) or a modified form of itAll new proteins begin with Met, although it isusually removed later by a protease
  80. 80. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe initiator tRNA is loaded into the P site ofribosome along with translation initiation factorsThe loaded ribosomal small subunit binds to the5’ end of the mRNA, recognized by the capThen moves forward along the mRNA searchingfor the AUGOnce found, large subunit associatesProtein synthesis begins with next tRNA bindingto the A site, etc.
  81. 81. Chapter 12: From DNA to Protein: Genotype to PhenotypeMechanism for finding start codon is different inbacteriaInstead of a 5’ cap, mRNA has specific ribosome-binding sequence located upstream of AUG =Shine-Dalgarno sequenceBacterial ribosome can also bind to this sequencewhen it is internal on the mRNA – importantdifference between procaryotes and eucaryotesNecessary for translation of polycistronic mRNAs– found only in bacteria
  82. 82. Chapter 12: From DNA to Protein: Genotype to Phenotype07_33_mRNA.encode.jpgRibosomes initiate translation at ribosome-binding sitesin polycistronic procaryotic mRNAs, which can encodemore than one protein**Note mistake in the legend to this figure in your text –Figure 7-33
  83. 83. Chapter 12: From DNA to Protein: Genotype to PhenotypeOne of three stop codons (UAA, UAG, UGA)signals the end of translationA protein release factor, rather than a tRNA,binds to a stop codonThis signals peptidyl transferase to add waterrather than an amino acid to the end of thegrowing polypeptideThis releases that last amino acid from the tRNA,and thus the polypeptide from the ribosomeThe ribosome releases the mRNA anddisassociates into its 2 subunits
  84. 84. Chapter 12: From DNA to Protein: Genotype to PhenotypeMost proteins begin folding into their 3-D shapeas they are being madeSome require molecular chaperones to help themfold correctly (review this term) – these bind to thepartially folded chain
  85. 85. Chapter 12: From DNA to Protein: Genotype to PhenotypeProteins are made on polyribosomes (orpolysomes)– several to many ribosomes spacedas close as 80 nucleotides along a single mRNA**Thus, many more proteins can be made in agiven time periodRemember too that translation is coupled totranscription in bacteria – both are going on at thesame time
  86. 86. Chapter 12: From DNA to Protein: Genotype to PhenotypeInhibitors of procaryotic protein synthesis areused as antibioticsThere are some important differences betweenprotein synthesis in bacteria v. eucaryotes, whichcan be exploitedWhy are these differences important in treatingbacterial infections?
  87. 87. Chapter 12: From DNA to Protein: Genotype to PhenotypeInhibitors of procaryotic protein synthesis areused as antibioticsThere are some important differences betweenprotein synthesis in bacteria v. eucaryotes, whichcan be exploitedWhy are these differences important in treatingbacterial infections?Need to be able to inhibit bacterial translation, butnot eucaryotic translation (or would be toxic tohumans)
  88. 88. Chapter 12: From DNA to Protein: Genotype to PhenotypeMany antibiotics are isolated from fungi! Why?
  89. 89. Chapter 12: From DNA to Protein: Genotype to PhenotypeNumber of copies of a protein in a cell dependson both how many are made, and how long theysurvive (like human population)**An important type of regulation on the amountof protein available in the cell is carefullycontrolled protein breakdowne.g. structural proteins may last for months oryears, enzymatic proteins for hours or secondsProteases act by hydrolyzing the peptide bondsbetween individual amino acids
  90. 90. Chapter 12: From DNA to Protein: Genotype to PhenotypeFunctions of proteolytic pathways:1) To rapidly degrade those proteins whoselifetimes must be short2) To recognize and eliminate proteins that aredamaged or misfolded (neurodegenerativediseases like Alzheimer’s, Huntington’s, andCreutzfeldt-Jacob disease are caused byaggregation of misfolded proteins)
  91. 91. Chapter 12: From DNA to Protein: Genotype to PhenotypeMost damaged proteins degraded in cytosol bylarge complexes of proteolytic enzymes calledproteasomesContain a central cylinder formed of proteaseswhose active sites face inwardCylinder is stoppered on ends by large proteincomplex – binds the proteins to be degraded,unfolds them, and then feeds them into cylinder,using ATP
  92. 92. Chapter 12: From DNA to Protein: Genotype to Phenotype07_36_proteasome.jpgThe proteasome degrades unwanted proteinscapcylinder
  93. 93. Chapter 12: From DNA to Protein: Genotype to PhenotypeProteasomes recognize proteins to be degradedby the attachment of a small protein calledubiquitinUbiquitin added to special amino acid sequences,or to abnormal amino acids or motifs that arenormally buried
  94. 94. Chapter 12: From DNA to Protein: Genotype to Phenotype07_37_Protein.produc.jpgAll of thesesteps can beregulated bythe cell
  95. 95. Chapter 12: From DNA to Protein: Genotype to PhenotypeRNA and the Origins of LifeOne view is that an RNA world existed on Earthbefore modern cells aroseIn primitive cells, RNA both1) stored genetic information2) catalyzed chemical reactionsEventually, DNA took over as genetic materialProteins became major catalysts and structuralcomponents
  96. 96. Chapter 12: From DNA to Protein: Genotype to Phenotype07_38_RNA world.jpg
  97. 97. Chapter 12: From DNA to Protein: Genotype to PhenotypeSome RNA catalysts carry out fundamentalreactions in modern-day cells= molecular fossils of an earlier worldFor example:ribosomesRNA splicing machineryThe arguments in support of the RNA worldhypothesis……..
  98. 98. Chapter 12: From DNA to Protein: Genotype to PhenotypeLife requires autocatalysisThe origin of life requires molecules with theability to catalyze the production of moremolecules like themselvesThese would out compete othersWhat molecules have autocatalytic properties?
  99. 99. Chapter 12: From DNA to Protein: Genotype to PhenotypeLife requires autocatalysisThe origin of life requires molecules with theability to catalyze the production of moremolecules like themselvesThese would out compete othersWhat molecules have autocatalytic properties?Best catalysts are proteins, but can’t reproducethemselves directly
  100. 100. Chapter 12: From DNA to Protein: Genotype to PhenotypeLife requires autocatalysisThe origin of life requires molecules with theability to catalyze the production of moremolecules like themselvesThese would out compete othersWhat molecules have autocatalytic properties?Best catalysts are proteins, but can’t reproducethemselves directly**But RNA can both store information andcatalyze reactions
  101. 101. Chapter 12: From DNA to Protein: Genotype to PhenotypeRNA can specify the sequence of acomplementary polynucleotide, which in turn canspecify the sequence of the original molecule
  102. 102. Chapter 12: From DNA to Protein: Genotype to Phenotype07_39_copy_itself.jpgRNA can make an exact copy of itselfResults in “multiplication” of the original sequence
  103. 103. Chapter 12: From DNA to Protein: Genotype to PhenotypeBut efficient synthesis also requires catalysts topromote fast, efficient, error-free reactionsToday, the protein RNA and DNA polymerases dothatWhat did it before proteins had appeared?Even today, have ribozymes with catalytic activity– what?
  104. 104. Chapter 12: From DNA to Protein: Genotype to PhenotypeBut efficient synthesis also requires catalysts topromote fast, efficient, error-free reactionsToday, the protein RNA and DNA polymerases dothatWhat did it before proteins had appeared?Even today, have ribozymes with catalytic activity– what?1) the rRNA that catalyzes the peptidyltransferase reaction on the ribosome2) the snRNAs in the snRNPs that catalyzesplicing
  105. 105. Chapter 12: From DNA to Protein: Genotype to PhenotypeA single-stranded RNA molecule can base-pair toitself (with both conventional and “non-conventional” hydrogen bonding, thus folding intocomplex 3-D structureThese too can act as catalysts, because of theirsurface with unique contours and chemicalpropertiesBut since have only 4 types of nucleotides, therange of chemical reactions, and efficiency, islimited
  106. 106. Chapter 12: From DNA to Protein: Genotype to Phenotype07_40_ribozyme.jpgRibozyme = anRNA moleculewith catalyticactiviites
  107. 107. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe processes in which catalytic RNAs play a roleare some of the most fundamental steps in theexpression of genetic information---**especially those steps where RNA moleculesthemselves are spliced or translated into proteins
  108. 108. Chapter 12: From DNA to Protein: Genotype to Phenotype
  109. 109. Chapter 12: From DNA to Protein: Genotype to PhenotypeThus, RNA has all the properties required of amolecule that could catalyze its own synthesisSelf-replicating systems of RNA molecules not yetfound in nature, but scientists believe they can beconstructed in the lab
  110. 110. Chapter 12: From DNA to Protein: Genotype to Phenotype07_41_catalyze_synt.jpgA hypothetical RNA molecule that could catalyze its ownsynthesis
  111. 111. Chapter 12: From DNA to Protein: Genotype to PhenotypeRNA is thought to predate DNA in evolutionEvidence that RNA arose before DNA found inchemical differences between them:1) Ribose is readily formed from formaldehyde(HCHO), one of principal products of experimentssimulating conditions on primitive earthDeoxyribose made from ribose, catalyzed by aprotein todayThus, suggestion that ribose came first
  112. 112. Chapter 12: From DNA to Protein: Genotype to PhenotypeOnce DNA appeared, it proved more suitable forpermanent storage of genetic information---1) It’s chemically more stable than RNA (becauseof the deoxyribose), so can maintain longerchains without breakage2) It’s double-stranded, so a damaged nucleotideon one strand can be easily repaired by usingthe other strand as template3) Using thymine rather than uracil makesdeamination easier to repair (deam. C → U)
  113. 113. Chapter 12: From DNA to Protein: Genotype to PhenotypeEventually in cells,DNA took over for information storageProteins took over as catalysts because ofgreater chemical complexityRNA remains as the intermediary connectingthemAnd cells could become ever more complex,evolving great diversity of structure and function
  114. 114. Chapter 12: From DNA to Protein: Genotype to Phenotype07_42_RNA_DNA.jpg
  115. 115. Chapter 12: From DNA to Protein: Genotype to PhenotypeHow We Know – Cracking the Genetic CodeResearchers began by perfecting the isolation ofa cell-free system that could synthesize proteinsfrom added synthetic RNAsCould only use polynucleotide phosphorylase atfirst, which randomly joined togetherribonucleotides present in the test tubeFirst tested poly-UUUUUUUU → phenylalanine
  116. 116. Chapter 12: From DNA to Protein: Genotype to Phenotype07_24_UUU codes.jpg
  117. 117. Chapter 12: From DNA to Protein: Genotype to PhenotypeAnd, poly-AAAAAAAAA → lysinepoly-CCCCCCCC → prolinepoly-GGGGGGG base-paired and didn’twork
  118. 118. Chapter 12: From DNA to Protein: Genotype to PhenotypeEventually figured out how to make mixedpolynucleotides, which were harder to interpret:e.g. UGUGUGUGUG → cysteine and valine, butwhich is which, since have both UGU and GUGcodons?
  119. 119. Chapter 12: From DNA to Protein: Genotype to Phenotype07_25_coding.jpg
  120. 120. Chapter 12: From DNA to Protein: Genotype to PhenotypeEventually figured out how to make RNAfragments only 3 nucleotides in lengthThese would bind to ribosomes and attract theappropriate charged tRNAHad only to to capture these on filter paper, andthen identify the attached amino acidWithin a year, the entire code was deciphered!
  121. 121. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe central dogmaDNA structureDNA replicationRNA structureRNA synthesis(Transcription)The genetic codeProtein synthesis(Translation)MutationConsequences of mutationLecture 1Lecture 2Lecture 3Lecture 4Topics
  122. 122. Chapter 12: From DNA to Protein: Genotype to PhenotypeMutationsMutation- change in DNA sequence leading toa different protein sequence being produced-same codon producedMissense- different codon introducedSilent (acceptable)Partially acceptableNonsense-stop codon introducedUsually unacceptable
  123. 123. Chapter 12: From DNA to Protein: Genotype to PhenotypeEnergeticsEach amino acid residue requires 4 ATPequivalentsATP AMP + PPi to “charge” tRNA1 GTP is used to place aminoacyl-tRNA intoA-site1 GTP is used to translocate after eachpeptide bond formation
  124. 124. Chapter 12: From DNA to Protein: Genotype to PhenotypeRegulation of Translation1. Elongation factor 2-a. phosphorylated under stressb. when phosphorylated, doesn’t allowGDP- GTP exchange and proteinsynthesis stops2. eIF-4E/4E-BP complex can bephosphorylated
  125. 125. Chapter 12: From DNA to Protein: Genotype to PhenotypePost-translational Modifications1. Proteolytic cleavage (most common)a. Direction into the ER and signal sequencecleavageb. Other signal sequences exist for otherorganellesc. Activation2. Disulfide bond formation
  126. 126. Chapter 12: From DNA to Protein: Genotype to PhenotypePost-translationalModifications, contd.3. Group additiona. Glycosylation (most complex known)b. Acetylation or phosphorylation, etc.4. Amino acid modificationa. Hydroxylation of Pro (in ER)b. Methylation of LysThis list is not exhaustive
  127. 127. Chapter 12: From DNA to Protein: Genotype to PhenotypeGenetic RegulationConstitutive vs. InducibleExpressionConstitutive- A gene is expressed at the samelevel at all times. AKA housekeeping gene.Inducible- A gene is expressed at higher levelunder the influence of some signal.
  128. 128. Chapter 12: From DNA to Protein: Genotype to PhenotypeGenetic Regulation - The OperonOperon- an operator plus two or more genes undercontrol of that operatorOccurs only in prokaryotes (in eukaryotes, eachgene is under separate control).Best known is the lac operon of Jacob and Monod
  129. 129. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe Operon Under NormalExpression
  130. 130. Chapter 12: From DNA to Protein: Genotype to PhenotypeThe Operon Under InducedExpression
  131. 131. Chapter 12: From DNA to Protein: Genotype to PhenotypeEukaryotic TranscriptionalRegulationTATA box- where to startCAAT box and Enhancer- how often to startEnhancer CAAT TATA Gene
  132. 132. Chapter 12: From DNA to Protein: Genotype to PhenotypePost-TranscriptionalRegulation1. mRNA stability can be altered by signalmoleculesPEPCK +Insulin = 30 min -Insulin = 3 h

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