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Basic Genetics

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Overview of Basic Genetics

Overview of Basic Genetics

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  • 1. MM Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 2. MM – DNA Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 3. BASENUCLEOTIDES PO4 CH2• Building blocks of DNA O• Made up of SUGAR • Pentose sugar • Nitrogen base (1’) OH • Phosphate group (5’)• Link to form sugar phosphate backbone of DNA • Phosphodiester bond b/w 3’ OH and 5’ PO 4 • Covalent bond
  • 4. NUCLEOTIDE BASES• 2 kinds of bases each with 2 types • Purine • Adenine (A) • Guanine (G) • Pyramidines • Thymine (T) • Cytosine (C)• Bases form hydrogen bonds to hold both strands • Bonds are complimentary and specific: purine with pyrimidine • A - - T (2 H bonds) • G - - - C (3 H bonds) • Hence both strands are complementary (reflections of each other)
  • 5. DNA – STRAND STRUCTURE• Made up of nucleotides• 2 strands • Each strand is made of • Sugar phosphate backbone on outside (because it is hydrophilic) • Formed by phosphodiester bonds b/w 3’ OH and 5’ PO4 • Bases protrude on inside of helix (because they are hydrophobic and H bond together) • Anti-parallel direction • Direction marked by free 5’ PO4 or 3’ OH group on the end • The 5’ of one strand is in front of the 3’ of the other • Complementary • One strand has the information
  • 6. DNA – HELIX STRUCTURE• 2 strands make a helix • Double helix • Wound around common axis • Right handed helix • Diameter = 20 A = 2 nm • Bases separated by 3.4 A and 30 o rotation • Helix has 2 groovs • Major Groove (22 A wide) • Bases more exposed  proteins bind DNA sequences here • Minor Groove (12 A wide)
  • 7. DNA – BONDS• Order of collective strength • Covalent bond • Phosphodiester bonds • Van der waals forces • Between bases on same strand • Hydrogen bond • Between bases on different strands
  • 8. DNA – MACROSTRUCTURE • 2 strands wrapped in double helix • Double helix wrapped around histones  beads on string • = sequence of nucleosomes (DNA + Histones) • Beads on string loops into a solenoid • Solenoid loops on itself supported by scaffold proteins  looped domains (interphase) • Looped domains loops around itself • This is packed into a chromosome (metaphase)
  • 9. DNA MACROSTRUCTURE – DEFINITIONS• Chromatin • DNA + protein• Chromosome • compacted chromatin• Chromatid • 1 of a duplicate of chromosome strands formed in cell division and separated in the last phase to become individual chromosomes • Duplication occurs in mitosis• Nucleosome • Sequence of DNA wrapped around one histone complex
  • 10. NUCLEOSOMES• DNA + Histones• Nucleosome involves 2 sets of 4 subtypes of histones • 2x H2A • 2x H2B • 2x H3 • 2x H4• Histones interact with DNA because they have a lot of +ve amino acids (Lysine) which interacts with –ve DNA• H1 attaches to linker DNA b/w neucleosomes
  • 11. CHROMATIN – CLASSIFICATION• 2 kinds • Euchromatin • Readily accessible DNA • Acetylation of bases  relaxation of DNA into euchromatin • Heterochromatin • Supercoiled and compacted • Not accessible • Methylation  compaction of DNA into heterochromatin • Some areas of DNA always in heterochromatin form
  • 12. CHROMOSOME – STRUCTURE• Compacted chromatin• Has centromere • Holds chromatids together • Attaches to mitotic spindles • Attaches to homologous chromosome• Has telomere • Repetitive DNA • Protects ends of chromosomes• Has 2 arms • Longer arm (p) • Shorter arm (q)
  • 13. CHROMOSOME – PROCESSING• Banding • Stain with Gimensa stain  light and dark bands • Dark bands (G bands) are heterochromatin • Light bands (R bands) are euchromatin• Karyotyping • Representing all chromosomes by • Number • Type • Shape
  • 14. DNA – PROCESSES• Denaturation • Separating DNA strands • Involved breaking of H bonds • Starts in A - - T rich areas • Causes: • Temperature (melting) • Melting Temperature: temperature at which 50% of DNA is denatured • High pH • Low salt• Renaturation (annealing) • Occurs if heat denatured DNA is cooled
  • 15. DNA – MODIFICATION• Methylation • Chemical modification • Adding methyl group to C • Makes DNA inactive • Makes structure inaccessible to proteins• Mutations • DNA sequence changed by mutagens  damages DNA • Mutagens • Radiation (X-ray / UV) • chemicals
  • 16. DNA – FUNCTION• Stores genetic information• 1 gene = information for 1 protein / RNA + its regulatory information• Gene is made of many codons • 1 codon = 3 nucleotides = information for 1 amino acid • Sequence of codons = sequence of amino acids in protein• Genome = sum total of all DNA in organism • Humans: 23 pairs of chromosomes, one pair is sexual• Human Genome Project = identify all genes of human genome
  • 17. MM – CENTRAL DOGMA Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 18. CENTRAL DOGMA (FLOW OF GENETIC INFO)Replication DNA Transcription RNA Translation PROTEIN Function • Problem in flow  • Cancer • Chronic illness • Mutation
  • 19. UNIQUE PROCESSES Reverse Transcription RNA (Viruses) DNA RNA Replication (Viruses & RNA Plants) RNA Protein Replication (Prions) Protein Protein
  • 20. MM – REPLICATION Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 21. DNA REPLICATIONReplication DNA Transcription RNA Translation PROTEIN Function • Problem in flow  • Cancer RNA • Chronic illness primer • Mutation
  • 22. DNA REPLICATION – REQUIREMENTS• Enzymes (Replisome) • Helicase • Primase • Polymerase: elongates primer  replicating DNA • Topoisomerase • Ligase: connects loose ends of DNA fragments• Proteins • ssBP (single stranded binding proteins) • Sliding clamp • Encircles DNA and binds polymerase  increase processivity• dNTPs + Mg2+• Single stranded template strand • Semiconservative
  • 23. DNA REPLICATION –PROCESS• Initiation• Priming• Elongation• Depriming• Ligating• Terminating
  • 24. DNA REPLICATION – PROCESS• Initiation • Starts at origin of replication (Ori) • Eukaryotes: many sites  many replication forks • Prokaryotes: one site  one replication fork • AT rich sequence • Separation of both strands • DNA Helicase unwinds helix • Requires ATP • ssBP bind to exposed bases to prevent reannealing • Topoisomerase • Uncoils supercoiled part of DNA
  • 25. DNA REPLICATION – PROCESS• Priming • Primase  RNA Primer • In eukaryotes it is a/w DNA pol a• Elongation • DNA polymerase elongates primer • Requires free 3’ OH group • Specific directionality • Reads: 3’ to 5’ • Makes new: 5’ to 3’ • Prokaryotes: DNA pol III • a/w Sliding Clamp • Eukaryotes: started by DNA pol a and continued by d • Pol d a/w Proliferating Cell Nuclear Antigen (PCNA)
  • 26. DNA REPLICATION – PRO: PROCESS• DNA Polymerase can only elongate in 5’ to 3’ direction• Both strands replicated simultaneously•  Semidiscontinuous Replication • Leading strand • Replicated continuously • Lagging strand • Replicated discontinuously in fragments (Okazaki Fragments) • Primase makes new primer at regular intervals • DNA Pol elongates it in 5’ to 3’ direction (NEW) • DNA Pol blocked when near new primer
  • 27. DNA Polymerase – ClassificationPOC Prokaryote EukaryoteDNA Pol I II III α β ε δ γ Locates nick Elongates Initiates repl’n b/w OF, primer, Completes a/w PrimaseFunctio Removes RNA catalyzing on pol a Mitochon- ahead, Repair Extends Repair Repair drial DNA n Replace with PDEB replicating primer by Leading and Replication DNA, Replace short piece of Lagging primer DNA DNAProofre ading YES N/A YES x YESPolyme 5’  3’ 5’  3’ 5’  3’ 5’  3’ 5’  3’ raseExonuclease 3’  5’ 3’  5’ 3’  5’ High: x 3’  5’Proces Sliding Moder High: sivity Clamp ate PCNA
  • 28. DNA REPLICATION – PROCESS• Depriming • Prokaryotes: Replacement of RNA primer by DNA pol I • Locates nick b/w OF  Removes RNA ahead  Adds DNA • Eukaryotes: • Rnase H1 removes RNA  FEN1 removes last RNA and proofreads forward 15 bp  DNA pol d copies into DNA• Ligating • Ligase connect loose ends of DNA
  • 29. DNA REPLICATION – PRO: PROCESS• Termination • Have termination sequences opposite to Ori • Proteins bind sequence  • Prevent helicase unwinding  • Dissociation of replisome • Eukaryotes • Terminate when replication forks collide • End of lagging strand (3’) filled with telomeres • TTAGGG tandem repeats • Synthesized by telomerase • RNA template for telomere • Normally in rapidly diving cells ex. Gametes • Function declines as cell develops  Telomere shortens  DNA damage  stop division • Absence  senescence; enhanced  Cancer
  • 30. DNA REPLICATION – PRO/EUK DIFFERENCES POC PRO EUK Initiation 1 Ori  1 fork Many Oris  many forksElongation DNA pol III DNA pol a  d RNA removed by Rnase H1  FENDepriming Replased by DNA Pol I 1 removes last 5’ RNA and proofreads 15 bp after  DNA Pol d makes DNA Termination sequences  bind Terminate when replication forksTermination protein  dislocate Helicase  end replication meet End of 3’ end filled with telomeres
  • 31. DNA REPLICATION – NOTES• Need to disassemble nucleosomes and reassemble• Random distribution of histones
  • 32. MM – DNA ERRORS, DAMAGE, AND REPAIR Androu Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
  • 33. DNA REPLICATION – ERRORS• Errors cause mutation if not repaired• Errors prevented • Substrate specificity • DNA Pol only catalyzes reaction between complementary bases • Proofreading• Errors repaired
  • 34. DNA DAMAGE• Constant• Agents • Radiation • Chemicals• Cell repairs damage• Causes mutations if not repaired • Insertion • Deletion • Substitution
  • 35. DNA REPAIR• 5 ways • Mismatch repair • Base excision repair • Nucleotide excision repair • Nonhomologous End Joining • Recombination Repair
  • 36. DNA REPAIR – MISMATCH REPAIR• Process • Mismatch  • Kink  • MutS binds  • MutL recruited  • DNA forms loop  • MutH breaks daughter strand (parent methylated)  • UvrD unwinds DNA  • Exonuclease removes DNA  • DNA pol makes DNA  • Ligase joins ends• Defect  HNPC (Heriditary Non Polyposis Cancer)
  • 37. DNA REPAIR – BASE EXCISION REPAIR• Process • Base lost chemically  • Removed by DNA glycosylase  • AP endonuuclease cuts backbone  • Exonuclease removes base  • DNA Pol makes DNA • Ligase joins ends
  • 38. DNA REPAIR – NUCLEOTIDE EXCISION REPAIR• Process • Kink in chain  • UvrABC endonuclease cleaves both sides  • UvrD removes sequence  • DNA Pol makes DNA • DNA Ligase joins ends• Defect  Xeroderma Pigmentosum (AR) • Photosensitivity • Sking CA
  • 39. DNA REPAIR – NHEJ• Process • Double stranded break  • Ku protein senses break  • Holds both strands  • Ends are aligned, trimmed, or filled  • DNA Ligase joins strands• Causes mutations• Deficiency  CA and Immunodeficiency Syndrome
  • 40. DNA REPAIR – RECOMBINATION REPAIR• Process • Double stranded break  • Recombination • Uses info of homologous chromosome to repair• Defect  Breast CA • Ex. BRCA 1 and BRCA 2
  • 41. MM – TRANSCRIPTION Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 42. TRANSCRIPTIONReplication DNA Transcription RNA Translation PROTEIN Function • Problem in flow  • Cancer • Chronic illness • Mutation
  • 43. TRANSCRIPTION - GENES [+1]Upstream Downstream-4-3 -2 P-1 CODING REIGON T RNA 5 3
  • 44. TRANSCRIPTION – GENERAL 5 GENE 1 GENE 3 3 3 5 3 5 3 GENE 2 5 5 3
  • 45. TRANSCRIPTION – REQUIREMENTS• Promoter on DNA • Conserved sequence • TATAAT• RNA Polymerase • No primer required • 4 subunits • α • β: Binds NTPs + Catalyze bond formation • β’: Binds DNA template • σ: recognizes promoter sequence• RNTPs : A, G, C, U
  • 46. TRANSCRIPTION – PROCESS• Initiation • RNAP binds promoter sequence ( σ) • Unwinds Promoter• Elongation • σ dissociates • RNA Polymerase reads ONE strand in 3’  5’ •  make unbranched RNA in 5’  3’ direction • RNA = Complementary strand •  Transcription bubble that moves along strand• Termination • Transcription of terminator sequence (3’UTR)  RNAP dissociate
  • 47. TRANSCRIPTION – TERMINATION• Terminator sequences • Hairpin loop • GC rich •  hairpin structure (stem and loop structure) • Followed by poly-U •  weak hybridization b/w DNA and RNA •  RNAP pauses  RNAP dissociates
  • 48. TRANSCRIPTION – PRODUCTS• Always RNA, usually single stranded, unbranched • tRNA • Involved in translation • tRNA genes • Not translated • rRNA •  ribosomes for translation • rRNA genes • Not translated • mRNA • Translated  protein • Protein coding genes
  • 49. TRANSCRIPTION – EUKARYOTES• 5 differences • Require regulatory proteins to expose promoters • DNA Packaging • RNA processing & exporting • Nucleus •  translation and transcription not simultaneous • Has 4 RNA Polymerases • RNAP I  rRNA (Nucleolus) • RNAP II  mRNA precursors (Nucleoplasm) • RNAP III  tRNA and 5S rRNA (Nucleoplasm) • Mitochondrial RNA Pol  mtRNAs (Mitochondrion) • More extensive transcription control • Post-transcriptional mRNA processing
  • 50. TRANSLATION (EUKS) – MRNA PROCESSING 1o DNA RNA Modified Pol II Transcript• Sum total of 1 o transcripts = heterogeneous nuclear RNA (hnRNA)• Modification • 5’ Cap • Splicing • 3’ poly(A) tail
  • 51. TRANSCRIPTION (EUKS) – 5’ CAPPING• 7-methyl-guanosine residue• 5’ tp 5’ triphosphate link• Guanyltransferase• Cap binds proteins • protect mRNA from nuclease • Guides mRNA export through nuclear pore • Initiation of transcription
  • 52. TRANSCRIPTION (EUKS) – SPLICING • Gene has coding sequences (exons) and non-coding sequences (intron) • Splicesome non-coding intron sequences • Done during transcription after 5’ capping before export 5’ Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 3’5’ m7GPPP Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 3’ Splicesome 5’ m7GPPP Exon 1 Exon 2 Exon 3 3’ Intron 1 Intron 2
  • 53. TRANSCRIPTION (EUKS) – SPLICESOME• Composed of • snRNA (Small Nuclear RNA) + • Proteins •  snRNPs (Small Nuclear Ribonucleoproteins)• Recognizes consensus sequences at ends of introns snRNA Proteins snRNP
  • 54. TRANSCRIPTION (EUKS) – 3’ TAIL• Process • Polyadenylation signal sequence from termination sequence (AAUAAA) • Recruit endonuclease  • Cleave 20 bases downstream of sequence • Poly(A) polymerase adds 40-250 A to cleaved end• Function • Bind PABP (Poly-A Binding Protein) • Stabilize molecule • Protects against 3’ exonuclease • Facilitates export of mRNA• Shortened in cytosol
  • 55. TRANSCRIPTION (EUKS) – VARIABILITY• Can make more proteins than genes encode • Alternative Splicing • 1o Transcript  splice variants (may be tissue specific) • process • Retains / skips exons • Retains / skips introns • Shift splice site  different exon size • RNA Editing • 1o Transcript  introduce new stop codon • Done by enzymes • Ex: deamination of C to U by Apolipoprotein B Deaminase
  • 56. TRANSCRIPTION (EUKS) – ALTERNATIVESPLICING
  • 57. TRANSCRIPTION – MEDICAL USES• Antibiotics can stop transcription • Rifampicin • Binds β sub-unit of prokaryotic RNAP  prevents elongation • Actinomycin D • Binds DNA  prevents unwinding  prevents initiation
  • 58. MM – GENES Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 59. GENES• 1 gene = information for 1 protein• Has promoter and terminator sequence (consensus sequence)• Composed of sequence of codons [+1]Upstream Downstream-4-3 -2 P-1 CODING REIGON T RNA 5 3
  • 60. GENES – GENETIC CODE• 1 codon  code 1 amino acid in protein sequence• 1 codon = 3 base pairs • Simple math• Code cracked by trial of all possible codes• Code is • Degenerate • 1 amino acid  more than 1 codon • Differ in 3 rd base • Non-overlapping (read in triplets from mRNA) • Open Reading Frames
  • 61. TRANSLATION – OPEN READING FRAMES• Open Reading Frame Reading frame 1 • Read in non-overlapping triplets A U G U U U AAA U G G U G A • Determined by start codon location start Phe Lys Trp Stop • Only one ORF has useful informatiaon Reading frame 2 A U G U U U AAA U G G U G A Cys Leu Asn Gly Reading frame 3 A U G U U U AAA U G G U G A Val Stop Start Val
  • 62. MM – REGULATION OF EXPRESSION Androu Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
  • 63. EXPRESSION REGULATION – PROK / EUK POC Prokaryote Eukaryote Gene Groups Independenttranscription (operons) Negative Positive Regulation (repressor  (Activator (tf)  promoter) Enhancer)
  • 64. EXPRESSION REGULATION – GENERAL • Only express whats required • Cancer • Inefficient • Cellular specialization • Done by transcription factors • Protein binds promoter and enhancer  gene expressionDNA Many bases5’ 3’ Enhancer Promoter Transcribed Region - TF binding site
  • 65. EXPRESSION REGULATION – TYPES• Constitutive • Always on • Proteins always required  Balance b/w protein synthesis and half life • Regulated by tf that are always on• Inducible • Need to be turned on Nucleus • Respond to environment • Ex GF • Regulated by inducible tf • Signal transduction  activate tf
  • 66. EXPRESSION REGULATION TYPES – INDUCIBLE 1. Extracellular cues: Hormones, Cytokines, Cell-cell interaction 2. Receptors – Cell surface Cell - Intracellular 3. Signal transduction New Proteins - ultimate goal: activate TFs 4. Nucleus
  • 67. EXPRESSION REGULATION – EUK• 5 levels • Chromatin Structure • Transcription Initiation • Transcript processing • mRNA stability • Translation Initiation
  • 68. EXPRESSION REGULATION (EUK) – CHROMATINSTRUCTURE• Remodel to gain access • Tight chromatin  no access for tf to bind • Req unwinding  acetylation• Histones have tails  interact with neighboring DNA  chromatin structure • Tails have + Lys  interact with neighboring DNA  condense DNA • Histone Deacetylases (HDACs) • Acetylated tails have – charge  looser structure  exposure • Histone Acetyl Transferases (HATs)
  • 69. EXPRESSION REGULATION (EUK) –TRANSCRIPTION INITIATION• Most imp• Depends on • Strength of promoter • Enhancer element • Interaction with other bound factors• 2 types of promoters • Basal promoter • Enhancer element Coding sequence
  • 70. EXPRESSION REGULATION (EUK) – BASALPROMOTER• Essential• Close to start site• Function • Locates start of gene • Induces low level of transcription • Higher if more tf binding sites• Binds basal tf  RNA pol II binds  transcription• 2 types • TATA box • strong (binds all alone) • TFIID and TBP  RNA pol II  pre-initiation complex • Closer to start site of transcription • CCAT box • weak (requires co-activators to bind) • Farther from start site
  • 71. EXPRESSION REGULATION (EUK) – ENHANCERELEMENT• Function • Binds specific transcription factors • Enhances expression • Allows tissue specificity
  • 72. EXPRESSION REGULATION (EUK) – TFS• Protein bind promoter  regulate transcription• 3 domains • DNA binding domain • Dimerization Domain • Transactivation domain • Drives transcription• If TF found in tissue  expression • Tissue specificity• Activated by environmental cues • Expression • Active • Bind ligand • Bind inhibitor • Localization • Phosphorylation
  • 73. EXPRESSION REGULATION (EUK) – TF CONTROL– LOCALIZATION: NFKB• NFkB• Tf  inflammatory genes• Binds NFkB sites in promoters• Process • Stimulus  • IkB phosphorylated + ubiquitinated  • IkB degraded  • Release NFkB  • Goes to nucleus  • Binds promoter
  • 74. EXPRESSION REGULATION (EUK) – TF CONTROL– STIMULI: STEROIDS• Steroids pass through membrane • Bind steroid receptors • Dimerization • Enter nucleus • Bind SRE (Steroid Response Element) • DNA unwound by HATs  Recruit basal promoter and RNA pol II  transcription
  • 75. EXPRESSION REGULATION (EUK) – POST-TRANSCRIPTION• Alternative splicing • Ex Calcitonin• miRNA (microRNA) • Non-coding RNA • Bind complementary mRNA • Down-regulate expression • Disease • Cancer: miRNA binding E2F mRNA (regulates proliferation)
  • 76. EXPRESSION REGULATION (EUK) – MRNASTABILITY• Determined by 3’UTR • Protector factors bind it• Degraded by endonuclease• Ex TfR on transferrin mRNA • Makes transferrin • Transports Fe • Has Iron responsive element in 3’ UTR: binds IRBP  protective • Fe Low: TfR stable • Fe High: TfR unstable• Ex poly(A) tail • Binds PABP  protection
  • 77. EXPRESSION REGULATION (EUK) –TRANSLATION INITIATION• Initiation factor • Active/inactive • Level• Ex. Insulin • High  phosphorylate eIF4E  inhibits it
  • 78. MM – TRANSLATION Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 79. TRANSLATIONReplication DNA Transcription RNA Translation PROTEIN Function • Problem in flow  • Cancer • Chronic illness • Mutation
  • 80. TRANSLATION – GENERAL• mRNA codons code for amino acid  protein• Eukaryotes and prokaryotes • Eukaryotes • Processed mRNA exported from nucleus • Translation in cytoplasm OR RER • Prokaryotes • Translation co-transcriptional• 1 ribosome  1 mRNA• 1 mRNA  Many ribosomes = polyribosome
  • 81. TRANSLATION - REQUIREMENTS• mRNA • template• tRNA • Carries amino acids to mRNA • Specific• rRNA • Structural AND functional role in ribosome• Ribosomal Proteins• Protein factors: All GTPases rRNA Proteins Ribosomes
  • 82. TRANSLATION REQ’S – TRNA• Clover leaf structure • One amino acid binding arm • One anti-codon arm • Has wobble pos’n  efficiency • 20 tRNA for 20 amino acids• Amino acid bound by aminoacyl-tRNA-synthase • Needs ATP • Bound tRNA = charged tRNA• Specific to amino acid • Done by shape of tRNA •  recognition by diff synthase
  • 83. TRANSLATION TRNA – WOBBLE
  • 84. TRANSLATION REQ’S – RIBOSOME• Made of 2 subunits • Named after sedimentation coefficient • Each subunit made of rRNA + Protein• 2 kinds • Eukaryotes • 80 S made of 40 S and 60 S • Prokaryotes • 70 S made of 30 S and 50 S• Function: translation of mRNA using tRNA• Clinical: Chloramphinecol binds 50S --| peptidyl transferase --| translation
  • 85. TRANSLATION REQ’S – RIBOSOME• Has 3 sites • A (Aminoacyl) site • Binds new tRNA • P (Peptidyl) site • Has the protein being formed • E (Exit) site • Deacylated tRNA• Has 2 centres • Peptidyl transferase centre • Where peptide bond formation catalyzed • Decoding centre • Ensures only complementary anti-codon tRNA are added
  • 86. TRANSLATION – PROCESS• 3 stages • Initiation • Elongation • Termination
  • 87. TRANSLATION – INITIATION• General • Start Codon: AUG  Met • Inserted by initiator tRNA • Euk: embedded in Kozak Sequence • Start codon recognition sequence • GCC AUG •  efficent recognition• Process • 5’ cap recognition • Assembly of initiation complex = 40 S + Met-tRNA • Scan mRNA 5’  3’ (ATP) • Recognition of start codon  • assembly of complete ribosome • Initiation complex at P site
  • 88. TRANSLATION – ELONGATION• EF1-GTP • Entry of aminoacyl-tRNA into A site  EF1• GTP hydrolyzed and Ef1 released • Peptide bond forms b/w aa’s • Peptidyltransferase EF 2 • Chain moves from P to A site• Ribosome moves 1 codon • Driven by EF2 + GTP • Hydrolysis • tRNA moved from A to P • Empty tRNA moves P  E  released  recycled
  • 89. TRANSLATION ELONGATION – PEPTIDE BOND
  • 90. TRANSLATION – TERMINATION• Ribosome  Stop Codon (A)• Recognised by tripeptide in release factor• Release factor (RF1) binds to A site  • GTP hydrolysis • disassembly of the tRNA-ribosome-mRNA complex and • release of nascent polypeptide
  • 91. POST-TRANSLATIONAL EVENTS• Protein folding •  required structure for function • 1o (sequence of aa) 2o (a helix/b sheets) 3o (3D) 4o structure (multinumeric)• Post-translational modifications •  modify function and position • Example • Glycosylation: secreted • Fatty acyl groups: membrane anchors• Protein targeting •  moves protein to location
  • 92. POST-TRANSLATIONAL EVENTS – TARGETING• Short sequences of aa  target protein to location • Secreted • Nuclear • Nuclear Localization Sequence (NLS) • Recognized by proteins in nuclear pores
  • 93. POST-TRANSLATIONAL TARGETING –SECRETORY PROTEINS• Made in RER• Signal sequence at N end• Hydrophobic •  binds RER membrane •  moves protein through RER membrane •  signal sequence cleaved •  concentrated internally •  move into Golgi in transport vesicles •  move to Plasma membrane in secretory vesicles • Secretory vesicle fuses with membrane  protein expelled
  • 94. POST-TRANSLATIONAL TARGETING –SECRETORY PROTEINS
  • 95. MM – BIOTECHNOLOGY Androu WaheebMost pictures from MM lecture series given in RCSI-Bahrain
  • 96. BIOTECH – ISOLATION OF DNA • Tissue Sample • Homogenize Tissue Detergent • Lyse Cells High Salt • Precipitate Protein Centrifuge • Remove Protein Salt + Alcohol • Precipitate DNA Water / Buffer • Redissolve DNA -80 o C • Store DNA (Stable)
  • 97. BIOTECH – ISOLATION OF RNA• Problems • Tissue Sample • RNA is unstable • Homogenize Tissue • Degraded by RNA nucleases Chaotropic • RNA nucleases are stable solution • Lyse Cells• Chaotropic Solution High Salt • Precipitate Protein • Salts Centrifuge • Denature proteins • Remove Protein • Ex. Guanidium hypochloride Salt + Alcohol• Convert to DNA and store DNA • Precipitate RNA Water / Buffer • Redissolve RNA Stringent Conditions • Store RNA
  • 98. BIOTECH – ISOLATION OF MRNA• Isolate RNA• Isolate with poly(T) resin • Binds to poly(A) tail
  • 99. BIOTECH – CDNA SYNTHESIS• cDNA = Complimentary DNA = made from mRNA • Isolate RNA • Isolate mRNA Reverse Transcriptase + RNase H • cDNA - - mRNA Hydrolyze rest of RNA • ss cDNA Terminal deoxynucleotidyl transferase • Poly C Cap Ligate Poly G adaptor • Primed cDNA DNA Polymerase + dNTPs • ds DNA
  • 100. BIOTECH – RECOMBINATION• Recombination: manipulation of DNA• Uses • DNA sequencing • Diagnosis • Gene-therapy • Protein production • Research
  • 101. • Tools • DNA Modifying enzymes • Restriction endonucleases • Cloning Vectors • Organisms • Hybridization • Blotting • DNA Sequencing • PCR
  • 102. BIOTECH RECOMBINATION – RESTRICTIONENDONUCLEASES• Enzyme• Cleaves both DNA strands at specific site • Recognition sites • Pallindromic • Read same both ways• 2 types • Leaves blunt ends • Leaves sticky ends • Advantage in DNA addition
  • 103. BIOTECH – RESTRICTION MAPPING• Identifies different DNA • Cut DNA into restriction fragments with Restriction Endonucleases • Different sequences have diff # of restriction sites  • Different fragment sizes • Separate by electrophoresis  • Separate different fragments based on size • Different sequence = different restriction map• Too many fragment size combinations  smear
  • 104. BIOTECH RECOMBINATION – CLONING• Fragment of DNA  Vector  Introduced into cells  Replicated  Copy DNA• Vector must have • Ori • Selectable marker • Multiple cloning sites
  • 105. BIOTECH CLONING – VECTORS• Plasmids • Autonomously replicate• Bacteriophage lambda• BACs • Bacterial Artificial Chromosomes • Replicate long DNA• YACs • Yeast Artificial Chromosome
  • 106. BIOTECH – HYBRIDIZATION• ss complementary DNA sequences at 50-60oC anneal autonomously • Attach probe labeled with fluorescent or radioactive tag RNA• Differentiates different DNA• 3 kinds • Southern • DNA • Northern • RNA • Western DNA
  • 107. BIOTECH HYBRIDIZATION – S. BLOTTING • DNA Restriction Endonuclease • Fragmented DNA Gel Electrophoresis • Separate fragments Alkaline Solution • Denatures DNA Transfer to blotting membrane Add unrelated DNA • Blocks blotting membrane Add probe • Hybridises with complementary DNA Wash + Visualise
  • 108. BIOTECH HYBRIDIZATION – S. BLOTTING• Detects variations in DNA sequences involving the restriction site • Create different size restriction fragment • Diff in length = RFLP (Restriction Fragment Length polymorphism)• Use: DNA Fingerprinting• Ex. SCA
  • 109. BIOTECH HYBRIDIZATION – N. BLOTTING• Identifies RNA presence•• Hybridize RNA with DNA probe Same process as Northern RNA DNA
  • 110. BIOTECH – REVERSE HYBRIDIZATION• Reverse N. Blot• DNA probe on a chip• RNA fluorescently labeled and added• Expressed DNA will hybridize with RNA  labelling  identification
  • 111. BIOTECH HYBRIDIZATION – ARRAYHYBRIDIZATION• Deposit many DNA samples into hybridization matrix• Probe all simultaneously• Use microarrays • Cloned DNA fragments spotted onto slide • Oligos made in situ to probe • Hybridize with target • Target is labeled • Wash after exposure • If see label  target there
  • 112. BIOTECH – GENE CHIP• Array hybridiztaion• Oligonucleotides synthesized in situ squentially
  • 113. BIOTECH – GENE AMPLIFICATION (PCR)• Exponential increase in copies of target• Requirements • Template • dNTP + Mg • 2 Oligonucleotide primers • Designed artificially • Know some of the required sequence • Mark borders of gene to be amplified • Thermostable Polymerase (taq) • Thermal Cycler
  • 114. BIOTECH PCR – PROCESS
  • 115. BIOTECH – PCR PRODUCTS• Amplified amount of target DNA• Analyze sample • After Amplification • Real Time • Add probe oligonucleotide with fluorescent reporter and quencher • Quencher stops reporter when close • Taq pol had 5’  3’ exonuclease • When amplifying, it removes tag  tag away from quencher  tag fluoresces
  • 116. BIOTECH – DNA SEQUENCING• Sanger dideoxy chain termination method  controlled interruption of polymerization• ddNTP’s don’t have 3’ and 2’ OH group  No phosphodiester bond  Chain termination• Process • 4 reaction beakers • Each has • Template • Primer • dNTP + Mg • DNA pol • 1 kind of ddNTPs • Allow replication  strand stops at each position with the ddNTP • Electrophorese to separate • Polyacrylamide gel  separates diff of 1 nucleotide
  • 117. BIOTECH – DNA SEQUENCING Automated Fluorescence DNA sequencing
  • 118. BIOTECH – AUTOMATION• Automated Flouresceent DNA sequencing• High throughput DNA sequencing • Mass spectrometer• DNA Chip • Allows synthesis of oligonucleotides in situ to probe target • Add 1 nucleotide at a time• Other high througput methods • Real Time PCR • Pyrosequencing