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Transcription

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. Transcription Overview.

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  1. 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

  2. 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.

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

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

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

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

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

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

  9. 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

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

  11. 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

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

  13. 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

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

  15. 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

  16. 3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template mRNA stability …UGGCAGACCUGACCA 3′ …UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA

  17. 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

  18. 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

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

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

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

  22. 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

  23. Gene expression summary Prokaryotes Eukaryotes DNA DNA transcription transcription mRNA pre-mRNA cytoplasm nucleus • capping • polyadenylation • splicing directly translated (even before being completely transcribed) protein mature mRNA • transport to cytoplasm • translation cytoplasm protein

  24. Ribosome • Large ribonucleoprotein structure • E. coli: 3 rRNAs, 52 proteins • Two subunits: large and small large subunit RNA small subunit protein

  25. Eukaryotic Translation • How does the ribosome find the correct start codon? • Small ribosome subunit binds 5′ cap • Scans to first AUG start codon stop codon mRNA AAAAAAAAA… 3′ coding region 5′ cap 5′ UTR 3′ UTR

  26. After finding start codon, use the genetic code: Shown as mRNA 5′ → 3′ the Genetic Code

  27. Mechanics of Translation • Translation requires: • mature mRNA • ribosome • tRNAs • amino acids • accessory proteins

  28. tRNA • Small RNAs (74-95 nt) made by transcription • Intramolecular base pairing • Anticodon complementary to mRNA codon anticodon

  29. tRNA • “Charged” by specific aminoacyl tRNA synthetase

  30. Initiation of Translation • Small ribosome subunit binds at start codon • Prokaryotes: Shine-Dalgarno sequence (RBS) • Eukaryotes: binds cap, scans mRNA 5′ AUG GAU GGG

  31. Met Initiation of Translation • First tRNA (Met, anticodon CAU) joins complex 3' 5' UAC mRNA 5′ AUG GAU GGG

  32. Met Initiation of Translation • Large ribosomal subunit joins 3' 5' UAC mRNA 5′ AUG GAU GGG

  33. Met Initiation of Translation • P site holds tRNA with first aa • A site open for next tRNA 3' 5' UAC mRNA 5′ A P AUG GAU GGG

  34. Initiation of Translation

  35. Met Asp Elongation • Next tRNA enters 3' 3' 5' 5' UAC CUA mRNA 5′ A P AUG GAU GGG

  36. Met Asp Met Elongation • Peptidyl transferase forms peptide bond • Amino acid released from tRNA in P site mRNA 5′ 3' 5' 3' 5' UAC CUA AUG GAU GGG

  37. Asp Met Elongation • Ribosome translocates one codon • First tRNA binds briefly in E site until translocation completes mRNA 5′ 3' 5' 3' 5' UAC CUA AUG GAU GGG

  38. Asp Met Gly Elongation • Process repeats • Next tRNA can then enter the empty A site A P 3' 5' CCC mRNA 5′ 3' 5' CUA AUG GAU GGG

  39. Elongation

  40. Lys Val Phe Val Asp Gly Gly Asp Met Ile Leu Leu Termination • Ribosome stops at stop codon • No matching tRNA • Release factor binds A Gln P RF 3' 5' GUC UUG CAG UAG

  41. Translation complex dissociates Termination Lys Val Phe Val Met Gly Asp Asp Gly Ile Leu Leu Gln 3' 5' GUC UUG CAG UAG RF

  42. Polyribosomes Next ribosome starts as soon as start codon is available N Ribosome AUG mRNA Stop C N Growing polypeptide RNA subunits released 3' 5' direction of ribosome movement 5' – 3' Released polypeptide

  43. Protein Synthesis Pathways • Free ribosomes • Ribosomes bound to RER

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