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Transcription

Transcription. the Central Dogma. DNA. RPE65 gene. transcription. gene expression. mRNA. translation. Protein. RPE65 protein. Transcription. Individual DNA regions (genes) copied to mRNA One DNA strand is template Single-stranded RNA produced. mRNA. template strand.

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  1. Transcription

  2. the Central Dogma DNA RPE65 gene transcription gene expression mRNA translation Protein RPE65 protein

  3. Transcription • Individual DNA regions (genes) copied to mRNA • One DNA strand is template • Single-stranded RNA produced mRNA template strand template strand template strand template strand

  4. Transcription Overview Un beau jour, je suis allé au marché pour acheter du pain. Il faisait chaud. Alors, j’ai acheté aussi un limonade. Il faisait chaud.

  5. Transcription overview gene • What do we call this strand? CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA

  6. What enzyme makes RNA? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA

  7. What direction is mRNA made? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA

  8. What direction is the template strand read? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA template strand transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 3’ 5’

  9. Which strand does the mRNA look like? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA 3’ 5’ transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 3’ 5’

  10. How do we know where to start and stop? Transcription overview CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG DNA 3’ 5’ transcription – RNA polymerase CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC mRNA 3’ 5’

  11. Transcription overview RNA polymerase synthesizes RNA 5′→ 3′ Starts at promoter, ends at terminator • How is the RNA actually made? translation protein NH3 COOH “upstream” “downstream” +1 start codon stop codon DNA coding region promoter terminator transcription start codon stop codon mRNA coding region 5′ 3′ 5′ UTR 3′ UTR

  12. Prokaryotic transcription Promoter: -10 and -35 sequences 5’ DNA 3’ -35 -10 +1 mRNA 5′ TTGACAT AACTGTA 5′ TATAAT ATATTA

  13. Prokaryotic transcription Promoter: -10 and -35 sequences 5’ DNA 3’ -35 -10 +1 mRNA TTGACAT TATAAT

  14. Prokaryotic transcription Initiation: RNAP sigma subunit (σ) binds -10 and -35 σ 5’ DNA 3’ -35 -10 +1

  15. Prokaryotic transcription Initiation: RNAP core (α2ββ’) binds sigma α2ββ’ “core” σ 5’ DNA 3’ -35 -10 +1

  16. Prokaryotic transcription Initiation: Promoter determines template strand and direction 5′ 3′ 3′ 5′ -35 -10 template strand for gene 1 -35 -10 template strand for gene 2

  17. Regulatory elements • Prokaryotes use operator sequences Operators Protein Transcription factors 5’ DNA 3’ -35 -10 +1 mRNA TTGACAT TATAAT

  18. Prokaryotic transcription Initiation: RNAP opens transcription bubble (helicase activity) σ 5’ DNA 3’ -35 -10 +1

  19. Prokaryotic transcription Initiation: RNAP begins mRNA synthesis at +1 σ 5’ DNA mRNA 3’ -35 -10 +1

  20. Prokaryotic transcription Initiation: Sigma released σ 5’ DNA mRNA 3’ -35 -10 +1

  21. Elongation: Prokaryotic transcription 5’ DNA 3’ -35 -10

  22. Elongation: Prokaryotic transcription terminator 5’ DNA 3’

  23. Replication Transcription Synthesize DNA Copy whole genome Copy both strands Need primer 5′→ 3′ Multiple enzymes • How are replication and transcription similar? • How are they different? • Synthesize RNA • Copy one gene • Copy one strand • No primer • 5′→ 3′ • Only RNA polymerase

  24. Eukaryotic transcription 3 RNA polymerases: RNA polymerase I – rRNA RNA polymerase II – mRNA RNA polymerase III – tRNA RNA polymerase II from yeast

  25. Eukaryotic transcription RNAP II recognizes: TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences Enhancers Transcription factors TFIIB TFIID +1 Sp1 hERRa1 CAAT GATA TATA box

  26. Eukaryotic transcription RNAP II recognizes: TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences +1

  27. Eukaryotic Transcription Termination • Different from Prokaryotes- No terminator! • RNA cleaved from transcription complex +1 AAUAAA AAUAAA

  28. RNA processing in eukaryotes promoter DNA introns exons primary transcript transcription transcription (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence transport to cytoplasm for translation transport to cytoplasm for translation

  29. methylated guanine “backward” 5′ to 5′ linkage Not encoded in DNA Capping enzyme Recognition by ribosome 5′ cap 5′ AGACCUGACCAUACC

  30. RNA processing in eukaryotes promoter DNA introns exons primary transcript transcription transcription (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence transport to cytoplasm for translation transport to cytoplasm for translation

  31. 3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template Important for: Export of mRNA Initiation of Translation Stability of mRNA …UGGCAGACCUGACCA 3′ …UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA

  32. RNA processing in eukaryotes promoter DNA introns exons primary transcript transcription transcription (nucleus) 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence transport to cytoplasm for translation transport to cytoplasm for translation

  33. Splicing • Most genes interrupted by introns • Introns removed after transcription • Exons spliced together 5’ cap AAAAAAAAA 3’ poly - A tail splicing splicing AAAAAAAAA final mRNA unbroken coding sequence

  34. Splicing snRNPs recognize exon-intron boundaries RNA + protein Cut and rejoin mRNA

  35. Splicing RPE65 mRNA in nucleus: 21,000 nt (14 exons) AAAAAAAAA splicing splicing AAAAAAAAA mature RPE65 mRNA in nucleus: 1,700 nt (8%)

  36. Splicing Alternative splicing: >1 protein from one gene 27,000 human genes, but >100,000 proteins

  37. Splicing Mutations affecting splicing can cause genetic disease: cystic fibrosis retinitis pigmentosa spinal muscular atrophy Prader-Willi syndrome Huntington disease spinocerebellar ataxia myotonic dystrophy Fragile-X syndrome Or produce genetic susceptibility to disease: lupus bipolar disorder schizophrenia myocardial infarction type I diabetes asthma cardiac hypertrophy multiple sclerosis autoimmune diseases elevated cholesterol

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