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