Overview of 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