Elongation phase: Transcription bubble

1. Elongation phase: Transcription bubble Separation of RNA-DNA duplex 50 nt/sec • unwound region 17 bp • RNA-DNA hybrid duplex  8 bp • Mistakes 1 per 104- 1 per 104 nucleotides

3. RNA Synthesis in Bacteria Start of Gene Several RNA Pol transcribing the same gene

4. In bacteria, the new RNA can be immediately translated

5. G•C A•U C•G C•G G•C C•G C•G G•C Termination of RNA Polymerization C Two mechanisms possible 1) Rho () protein independent termination stable hairpin formation 2) Rho () protein-mediated termination U G U G A•U•U•U-3' 5'-U•C•C•C•A•C

6. -independent termination

7. -dependent termination

8. Antibiotic inhibitors of RNA polymerization Rifampicin inhibits initiation by blocking a channel within RNA polymerase: Actinomycin intercalates in ds DNA, inhibiting strand separation: phenoxazone

9. RNA synthesis in Eukaryotes See Stryer 5th edition p. 792-798 Or 4th edition p.

10. Eukaryotic vs prokaryotic transcription Prokaryotes: • no membrane-bound nucleus • transcription and translation are coupled Eukaryotes: • DNA is located in membrane-bound nucleus • Transcription and translation are separated in space and time

12. a-amanitin RNA Pol II Inhibitor Amanita phalloides (the death cap)

13. Actinomycin D •Antitumor antibiotic from Streptomyces genus • Aromatic ring intercalates between GC base pairs, while the peptides bind to the minor groove • Binds to GpC sequences in double-stranded DNA, stabilizing the duplex and inhibiting transcription • Inhibitor of eukaryotic RNA Pol I

14. Eukaryotic RNA Polymerase II • RNA Pol II is responsible for transcription to pre-mRNA • - 8-12 subunits • - Two large subunits are responsible for RNA synthesis • - Regulated by phosphorylation of carboxyl-terminal domain (CTD) of the largest subunit • - Unphosphorylated form is involved in initiation and phosphorylated form in elengation • - some subunits are shared for RNA Pol I-III

15. Type II Eukaryotic Promoters -25 +1 -90 -70 5' CAAT Box 3' GC Box TATA box (TATAAAA) (GGNCAATCT) (GGGGCG) Coding sequence Promoter sequence Start site Consensus sequence: Examples:

16. Initiation of transcription in eukaryotes • RNA Pol II can not initiate transcription by itself • Transcription factors (TFII) are required • The key initiation step is the recognition of TATA box by TBP

17. TATA box-binding protein (TBP)

18. Hydrogen bond donors and acceptors on each edge of a base pair

19. Formation of RNA Polymerase II pre-initiation complex IID contains TBP that binds TATA box IIA stabilizes IID binding to promoter IIB binds initiation sequence Pol II binds IIB IIE stimulates transcription IIH has kinase and helicase activity

21. Upstream elements (CAAT, GC) Proximal elements (TATA box) Enhancers Coding region Regulated expression Basal promoter Transcriptional control in eukaryotes

22. Enhancer sequences examples in eukaryotes

23. Post-transcriptional modifications of mRNA in eukaryotes • 5’ end CAP • polyA tail • splicing

24. Eukaryotic mRNA is 5’-Capped

25. PolyA Synthesis and termination of transcription

26. RNA splicing in eukaryotes Primary transcript, hnRNA

27. RNA Synthesis: Take Home Message 1) DNA sequences are translated into RNA messages by RNA polymerases. 2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences. 3) The synthesis of RNA is governed by initiation, elongation, and termination steps. 4) Eukaryotic mRNA is extensively processed