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Transcription. دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران. Dr. Parvin Pasalar Tehran University of Medical Sciences. Objectives: To know and explain: What are needed for transcription Template Building blocks Enzymes Energy Protein Factors Different Stages of transcription

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  1. Transcription دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران Dr. Parvin Pasalar Tehran University of Medical Sciences

  2. Objectives: • To know and explain: • What are needed for transcription • Template • Building blocks • Enzymes • Energy • Protein Factors • Different Stages of transcription • Initiation (promoter recognition) • Elongation • Termination • Differences between transcription in eu and prokaryotes • Toxins and antibiotics that inhibit transcription

  3. Transcription in Prokaryotes

  4. DNA Transcription Ribosome mRNA Translation Polypeptide (protein) Prokaryotic Transcription Cell

  5. What are needed for transcription • Permission • Template (DNA, Gene) • Building blocks (4 types of NTPs) • Enzymes • Energy (ATP and the other NTPs) • Protein factors (σ & ρ)

  6. Transcription Start Site 5’ 3’ Protein Coding Region 3’ 5’ RNA Transcript Promoter/ Control Region Terminator Sequence A “Simple” Prokaryotic Gene 3’ 5’ AUG UAA

  7. Promoter & itsconsensus sequence determination

  8. E. coli RNA polymerase • Holoenzyme: 2 ’  for initiation • Core enzyme: 2 ’ for elongation

  9. Different stages of Transcription • Recognition & Initiation • Elongation • Termination

  10. Initiation: close and open complex

  11. The elongation stage

  12. RNA chain termination Inverted repeats Termination occurs at terminator DNA sequences. 1. Rho-independent: the most common stop signal by RNA hairpin formation(self-complement structure) commonly GC-rich to favor the structure stability 2. Rho-dependent Termination: RNA wraps around Rho hexamer and stops transcription. ρ- independent ρ- dependent

  13. RNA Pol. RNA Pol. RNA 5’ RNA 5’ TerminationRho Independent Terminator

  14. RNA Pol. RNA Pol. 5’ RNA Terminator RNA 5’ TerminationRho Independent

  15. RNA Pol. r r RNA Pol. RNA 5’ Terminator RNA 5’ TerminationRho Dependent The terminator sequence slows RNA polymerase

  16. RNA Pol. r Help, rho hit me! RNA Pol. RNA 5’ r Terminator RNA 5’ TerminationRho Dependent Rho catches up with RNA polymerase

  17. RNA Pol. r r RNA Pol. 5’ RNA Terminator RNA 5’ TerminationRho Dependent The elongation complex disintegrates

  18. Transcription in Eukaryotes

  19. A “Simple” Eukaryotic Gene

  20. Cytoplasm Nuclear pores AAAAAA AAAAAA DNA Transcription RNA RNA Processing G G mRNA Export Nucleus Eukaryotic gene

  21. Different Eukaryotic RNA polymerases Basis of classification: 1- Elution at different salt concentrations during ion-exchange chromatography 2- Different sensitivity to α amanitin Pol I:very insensitive to α amanitin, transcribes the gene for pre-rRNA (28S, 5.8S, 18S) Pol II:very sensitive to α amanitin, transcribes all protein coding genes and the gene for snRNA Pol III:Intermediate sensitivity to α amanitin, transcribes the gene for tRNA, 5S rRNA, U6, RNA component of SRP

  22. Transcription factors Definition: Proteins other than purified RNA polymerase subunits, involved in Initiation, Elongation or Termination of transcription Classification: A: General transcription factors (GTFs): 1- are needed for basal gene expression 2- are required for RNA polymerase to bind avidly and specifically to the promoters. 3-GTFs for RNA polymerase II are called TFIIA , TFIIB , TFIID , … 4- can have multiple subunits B: Specific transcription factors: They are used for fine tune control of gene expression of specific genes

  23. RNAP II termination and poly adenylation

  24. mRNA modification The modifications signal the mRNA is ready to move out of the nucleus and may control its life span in the cytoplasm • 1) 5’ cap: modified guanine; protection; recognition site for ribosomes • 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport • 3) RNA splicing: exons (expressed sequences) kept, introns (intervening sequences) spliced out; spliceosome • 4) Alternative Splicing • 5) RNA editing

  25. Mechanism of RNA Splicing

  26. Alternative splicing Calcitonin gene-related peptide

  27. Toxins and antibiotics that inhibit transcription • Actinomycine D: makes cross link between the two strands of DNA, preventing it from being template for transcription • Rifamycine ( Rifampin): inhibits beta subunit of RNAP of prokaryotes • Ricin: A toxin from plant that inhibits RNAP of eukaryotes • Amanitin: A toxin from mashroom that inhibits RNAP of eukaryotes

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