replication (cont.)

Providing Great Care… Building Warrior Medics!

Replication (cont.)

15N

14N

Meselson, Stahl - 1958


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


Replication (cont.)

Replicons Prokaryotes Eukaryotes

Replisome components DnaB, DnaG, Pol III, SSBs

Semi-discontinuous Synthesis Leading and lagging Okazaki fragments Ligase


Replication (cont.)


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)


Transcription


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


Transcription (cont.)

4 phases Initiation

Elongation

Termination

Post-transcriptional

mods



Transcription Bubble Melt Dock/Initiate Extend Close

Transcript unstable 5’-methylated guanosine cap Poly-A tail Protects against

exonucleases Transcript transported outside

nucleus

SENSE doesn’t make sense



Post-transcriptional modifications

Keeping “tasty bits” Exons - Conserved

Selective pressure

Introns - Not conserved Lack of selective pressure

Remove these!



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?


Translation

Translation (cont.)


Translation


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!

Translation (cont.)


Translation (cont.)

Each codon has matched tRNA

Amino acid attaches to tRNA

Translation (cont.)


Translation (cont.)

4 phases Initiation

Elongation

Termination

Post-translational mod?

Amino acid structure

+NH3-CH[R]-COO-


Translation (cont.)


Translation (cont.)

Structural levels Primary

Secondary

Tertiary

Quaternary


Translation (cont.)


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

replication (cont.)

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