Basic Genetics
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This document provides information about DNA replication, transcription, and repair. It discusses the key components and processes of DNA replication including initiation, priming, elongation, and termination. It describes the central dogma of molecular biology involving the flow of genetic information from DNA to RNA to protein. The document also summarizes the main types of DNA repair including mismatch repair, base excision repair, nucleotide excision repair, nonhomologous end joining, and recombination repair.
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Basic Genetics
- 1. MM Androu Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
- 2. MM – DNA Androu Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
- 3. BASE NUCLEOTIDES 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 Waheeb Most 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 Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
- 21. DNA REPLICATION Replication 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 – Classification POC Prokaryote Eukaryote DNA Pol I II III α β ε δ γ Locates nick Elongates Initiates repl’n b/w OF, primer, Completes a/w Primase Functio 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 DNA Proofre ading YES N/A YES x YES Polyme 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ rase Exonu clease 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 forks Elongation DNA pol III DNA pol a d RNA removed by Rnase H1 FEN Depriming 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 forks Termination 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 Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
- 42. TRANSCRIPTION Replication 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) – ALTERNATIVE SPLICING
- 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 Waheeb Most 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 Independent transcription (operons) Negative Positive Regulation (repressor (Activator (tf) promoter) Enhancer)
- 64. EXPRESSION REGULATION – GENERAL • Only express what's required • Cancer • Inefficient • Cellular specialization • Done by transcription factors • Protein binds promoter and enhancer gene expression DNA Many bases 5’ 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) – CHROMATIN STRUCTURE • 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) – BASAL PROMOTER • 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) – ENHANCER ELEMENT • 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) – MRNA STABILITY • 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 Waheeb Most pictures from MM lecture series given in RCSI-Bahrain
- 79. TRANSLATION Replication 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 Waheeb Most 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 – RESTRICTION ENDONUCLEASES • 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 – ARRAY HYBRIDIZATION • 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