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Central Dogma

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  1. Recall Replication is semi-conservative and bidirectional transcription translation Central Dogma DNA RNA Protein replication Discussing DNA replication (Nucleus of eukaryote, cytoplasm of prokaryote)

  2. Lecture 10 DNA Replication *Leading and lagging strand synthesis Biochemistry of replication DNA mutation and repair Transcription

  3. 3’ end template ECB 6-10 5’ end DNA polymerase -adds nuclotides at 3’ end of existing strand 3’ OH Incoming nucleotide (triphosphate) adds at 3’OH of growing chain (condensation rx driven by cleavage of PiPi) Synthesis occurs in 5’ - 3’ direction Specificity of which base adds depends on base pairing with template strand ( strands are complementary)

  4. Problem: Strands are antiparallel; DNA made only 5’ to 3’ ECB 6-11 Fork moves at rate of ~1000 nucleotides/sec in prokaryotes (~100 NTs/sec in humans)

  5. Leading and lagging strands Replication fork is asymmetrical

  6. Lagging strand synthesis occurs from RNA primer ECB 6-16

  7. Helicase unwinds DNA duplex; breaks H bonds; requires ATP Single stranded binding proteins coat strand and prevent renaturation Sliding clamp keeps DNA polymerase bound Other Proteins at Replication Fork 06.5-DNA_replication_fork.mov ECB 6-17 Leading strand Lagging strand

  8. Lecture 10 DNA Replication Leading and lagging strand synthesis Biochemistry of replication *DNA mutation and repair Transcription

  9. ECB 6-19 Mutation: a permanent change in DNA sequence

  10. Mutations accumulate with age and cause cancer Cancer = loss of control of the cell cycle Incidents of cancer per 100,000 women ECB 6-20 Age (years)

  11. Cell mechanism to reduce: proofreading Cell mechanism to reduce: Many mechanisms of repair Mutations and Repair During Replication: Incorrect nucleotide incorporated Post Replication: Many sources of DNA damage

  12. Mistakes during replication cause mutations ECB 6-21

  13. 3’ end 5’ end Proofreading DNA Polymerase polymerase 3’ OH -Catalyzes phosphodiesterbond formation -Has exonuclease activity Incoming deoxy-ribonucleoside triphosphate -Performs proofreading 5’ end

  14. 3’ 5’ G 5’ A Proofreading mechanism T Exonuclease activity removes the incorrect nucleotide Polymerase activity then adds correct nucleotide After proofreading, mistakes about 1/107 nucleotides See ECB 6-13

  15. Replication 5’ Triphosphate Replication 3’ OH PP Proofreading requires 5’ to 3’ DNA synthesis Energy from pyrophosphate Energy from PP At 5’ end Error Error Incorrect dNTP removed Incorrect dNTP removed No PPP left At 5’ end 3’ OH available Correct dNTPbut cannot beadded Energy frompyrophosphate Corrected! ECB 6-15

  16. Mutations and Repair During Replication: Incorrect nucleotide incorporated Post Replication: Many sources of DNA damage Causes of Errors: Deamination Depurination Pyrimidine dimers Post-replication repair also removes 99% of errors made in replication; Final error rate 1/109 NT Cell mechanism to reduce: proofreading Cell mechanisms to reduce: Many, involve removing damaged DNA

  17. Post-replication repair requires excision, resynthesis, ligation How damaged strand is recognized is not understood ECB 6-26

  18. Lecture 10 DNA Replication Semi-conservative replication Biochemistry of replication End replication problem DNA mutation and repair *Transcription

  19. transcription translation Central Dogma DNA RNA Protein Nucleus of eukaryote Cytoplasm of prokaryote replication ECB 7-1

  20. Transcription control regions 5’ 3’ Coding region 3’ 5’ What is a gene? Double stranded DNA Protein A Protein B Protein C “each gene contains the information required to make a protein”

  21. How much of genome is composed of genes? Genome Projects: Bacteria - about 500 genes, most of genome Eukaryotes - about 20,000-40,000 genes, represents much less of genome Humans - about 30,000 genes,only a few percent of the total genome!!! Rest is repetitive DNA sequences - junk DNA Much of repetitive DNA is transposable elements that have mutated and can no longer move 15% of human genome is the L1 element 11% is Alu sequence, about 300 nucleotide pairs

  22. Single stranded Variety of 3D structures Ribonucleotides AUCG Organized as RNA-proteincomplexes DNA Structure: Double stranded Double helix Deoxyribonucleotides ATCG Organized as chromatin RNA Structure:

  23. RNA Structure ECB 7-3

  24. DNA codes for RNA Single-stranded product (5’ to 3’) Not H bonded to DNA RNA Polymerase Coded by DNA template strand (also called antisense strand)

  25. DNA template A:U two H bonds New NTP Base pairing Rate ~ 30 NT/sec

  26. 4 major RNA classes

  27. RNA Polymerases Prokaryotes: One RNA Polymerase, composed of four subunits, plus additional factors that can confer promoter specificity Eukaryotes: Three RNA Polymerases (RNA Pol I, II, III), each composed of >10 different proteins, transcribe different types of genes. RNA Pol. I: synthesizes ribosomal RNA (rRNA) RNA Pol II: synthesizes mRNA (protein coding) and some small RNAs. RNA Pol III: synthesizes a variety of small RNAs, including tRNAs.

  28. Lecture 10 DNA Replication Semi-conservative replication Biochemistry of replication End replication problem DNA mutation and repair Transcription-general *Prokaryotes Eukaryotes

  29. Prokaryotic Gene Organization Most genes in Operons: genes organized together, with one shared transcription start site One mRNA codes for Several proteins ECB 8-6

  30. Promoter: “nucleotide sequence in DNA to which RNA polymerase binds and begins transcription.” ECB definition Transcription of one strand Transcription Initiation in Bacteria - overview RNA Polymerase contacts DNA, slides along strand, “looking” for promoter sequences. At the promoter, the RNA Polymerase binds tightly, opening up a small single stranded region.

  31. Sigma subunit - binds RNA Pol., recognizes DNA sequencesin the promoter approximately 35 and 10 bases ‘upstream’ of transcription start site +1 = transcription start site Transcription Initiation in Bacteria (cont’d) ECB 7-9

  32. Initiation and termination of prokaryotic transcription ECB 7-9

  33. ECB 7-9 Terminator (stop sequence) Transcription termination in bacteria RNA Hairpin loop causes polymerase to fall off DNA

  34. Genome of Mycoplasma genitalium One of smallest genomes of any cell: codes for 470 proteins How does the cell know where to transcribe? … and when to transcribe? Proteins bind to specific DNA sequences, Some activate transcription, some repress Termed transcription factors

  35. Operator sequence associated with promoter Gene regulation in prokaryotes ECB 8-6 Regulatory protein (transcription factor) binds operator

  36. Repressible operon Default state is on, can turned off ECB 8-7

  37. Inducible operon; lac operon Default state is off, turned on