Download Molecular for Dummies part 2

Replication (cont.)
Meselson, Stahl - 1958
14N
15N
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Replication (cont.)
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Replication Fork
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Prokaryotes
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Eukaryotes
Ingredients
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Replisome
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dNTPs
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Template ssDNA
Energy to form bond
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Hydrolysis of extra
phosphates
Add 5’ to 3’-end of hanging
hydroxyl
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Replication (cont.)
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Replicons
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Prokaryotes
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Eukaryotes
Replisome components
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DnaB, DnaG, Pol III, SSBs
Semi-discontinuous Synthesis
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Leading and lagging
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Okazaki fragments
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Ligase
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Replication (cont.)
Providing Great Care… Building Warrior Medics!
Replication (cont.)
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Why is replication important to a lab tech?
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Mutations
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Point mutations - nonsense, missense, silent
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Transitions (G:C to A:T)
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Transversions (A:T to T:A)
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Sickle-cell disease - due to missense point mutation in
beta Hgb gene (valine inserted over glutamic acid)
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Base mispairing (e.g., A:C?)
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Deletions/Insertions (e.g., transposons)
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Transcription
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Transcription
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Proteins aren’t made in nucleus
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Need transition medium for passing information
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Messenger RNA (mRNA) to rescue!
Differences in DNA and RNA
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Pentose used  Ribose (2’ carbon)
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Pyrimidine used  Uracil vs. Thymine
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Single-stranded; can bond with itself
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Classes: mRNA, rRNA, tRNA, RNAi
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Transcription (cont.)
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4 phases
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Initiation
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Elongation
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Termination
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Post-transcriptional
mods
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Transcription (cont.)
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Transcription Bubble
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Melt
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Dock/Initiate
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Extend
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Close
Transcript unstable
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5’-methylated guanosine cap
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Poly-A tail
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Protects against
exonucleases
Transcript transported outside
nucleus
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SENSE doesn’t make sense
Transcription (cont.)
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Post-transcriptional modifications
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Keeping “tasty bits”
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Exons - Conserved
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Selective pressure
Introns - Not conserved
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Lack of selective pressure
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Remove these!
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Transcription (cont.)
Transcription (cont.)
Transcription (cont.)
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Why is transcription important to lab techs?
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Mutations!
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Pathology can erupt all along Central Dogma
Natural background mutation rate
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Repair mechanisms
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Molecular basis of evolution
Mutations can be advantageous or problematic
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Sickle-cell disease
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Cystic fibrosis
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Mutations that cause HIV resistance?
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Translation
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Translation
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Translation (cont.)
Translation
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Translation
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How is protein made from mRNA?
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Protein synthesis  ribosome
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mRNA transcript contains codons
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3-base triplet synonymous for certain
amino acid
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ORF (open reading frame)
Each amino acid may have >1
corresponding codon
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Other way around? NO!
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Translation (cont.)
Translation (cont.)
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Each codon has matched
tRNA
Amino acid attaches to
tRNA
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Translation (cont.)
Translation (cont.)
Translation (cont.)
Translation (cont.)
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4 phases
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Initiation
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Elongation
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Termination
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Post-translational mod?
Amino acid structure
 +NH3-CH[R]-COO-
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Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Translation (cont.)
Translation (cont.)
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Structural levels
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Primary
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Secondary
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Tertiary
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Quaternary
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Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Providing Great Care… Building Warrior Medics!
Translation (cont.)
Translation (cont.)
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Why is translation important to lab techs?
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Protein basis for many traditional lab assays
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Protein electrophoresis
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Hemoglobinopathies
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Sickle (quality)
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Thalassemias (quantity)
Enzyme kinetics assays
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CK, LDH, lipase, amylase, etc.
Colorimetric assays
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Total protein, albumin
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Lipoproteins
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All proteins have genetic origin
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Errors in translation/post-translation mods
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