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Replication (cont.)

Replication (cont.). 14 N. 15 N. 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

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Replication (cont.)

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  1. Replication (cont.) 14N 15N Meselson, Stahl - 1958

  2. 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

  3. Replication (cont.) • Replicons • Prokaryotes • Eukaryotes • Replisome components • DnaB, DnaG, Pol III, SSBs • Semi-discontinuous Synthesis • Leading and lagging • Okazaki fragments • Ligase

  4. Replication (cont.)

  5. 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)

  6. Transcription

  7. 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

  8. Transcription (cont.) • 4 phases • Initiation • Elongation • Termination • Post-transcriptional mods

  9. 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

  10. SENSE doesn’t make sense

  11. Transcription (cont.) • Post-transcriptional modifications • Keeping “tasty bits” • Exons - Conserved • Selective pressure • Introns - Not conserved • Lack of selective pressure • Remove these!

  12. Transcription (cont.)

  13. Transcription (cont.) Polymerase mRNA

  14. 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?

  15. Translation

  16. Translation Polymerase Ribosome mRNA

  17. Translation (cont.)

  18. Translation Codons Ribosome

  19. 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!

  20. Translation (cont.)

  21. Translation (cont.) • Each codon has matched tRNA • Amino acid attaches to tRNA

  22. Translation (cont.) Tertiary Secondary Primary?

  23. Translation (cont.)

  24. Translation (cont.)

  25. Translation (cont.) • 4 phases • Initiation • Elongation • Termination • Post-translational mod? • Amino acid structure • +NH3-CH[R]-COO-

  26. Translation (cont.)

  27. Translation (cont.) A A E E P P

  28. Translation (cont.)

  29. Translation (cont.) • Structural levels • Primary • Secondary • Tertiary • Quaternary

  30. Translation (cont.)

  31. Translation (cont.)

  32. Translation (cont.)

  33. Translation (cont.)

  34. Translation (cont.)

  35. Translation (cont.)

  36. Translation (cont.)

  37. Translation (cont.)

  38. 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

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