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

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

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

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

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

7. 7 Promoter & itsconsensus sequence determination Promoter & its consensus sequence determination

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

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

10. 10 Initiation: close and open complex

11. 11 The elongation stage

12. 12 RNA chain termination 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. Inverted repeats ρ- independent ρ- dependent

13. 13 RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

14. 14 RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

15. 15 RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  The terminator sequence slows RNA polymerase

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

17. 17 RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  The elongation complex disintegrates

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19. 19 A “Simple” Eukaryotic Gene

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

21. 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. 22 Transcription factors Definition: Proteins other than purified RNA polymerase subunits, involved in Initiation, Elongation or Termination of transcription Proteins other than purified RNA polymerase subunits, involved in Initiation, Elongation or Termination of transcription Classification: 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. 23 RNAP II termination and poly adenylation

24. 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 1) 5’ cap: modified guanine; protection; recognition site for ribosomes 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport 2) 3’ tail: poly(A) tail (adenine); protection; recognition; transport 3) RNA splicing: exons (expressed sequences) kept, introns (intervening sequences) spliced out; spliceosome 3) RNA splicing: exons (expressed sequences) kept, introns (intervening sequences) spliced out; spliceosome 4) Alternative Splicing 4) Alternative Splicing 5) RNA editing 5) RNA editing

25. 25 Mechanism of RNA Splicing

26. 26 Alternative splicing Calcitonin gene-related peptide

27. 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 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 Rifamycine ( Rifampin): inhibits beta subunit of RNAP of prokaryotes Ricin: A toxin from plant that inhibits RNAP of eukaryotes Ricin: A toxin from plant that inhibits RNAP of eukaryotes Amanitin: A toxin from mashroom that inhibits RNAP of eukaryotes Amanitin: A toxin from mashroom that inhibits RNAP of eukaryotes