1. Transcription

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

3. Transcription  Individual DNA regions (genes) copied to mRNA  One DNA strand is template  Single-stranded RNA produced template strand mRNA 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. mRNA DNA transcription CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG gene What do we call this strand? Transcription overview

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

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

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

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

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

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

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

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

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

15. Prokaryotic transcription Initiation:  RNAP core (α 2 ββ’) binds sigma DNA -35-10+1 σ α 2 ββ’ “core” 5’5’ 3’3’

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

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

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

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

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

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

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

23. ReplicationTranscription  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 +1+1 Enhancers Transcription factors Sp1 hERR  1 CAATGATATATA box TFIIBTFIID

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

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

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

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

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

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

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 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 fibrosisretinitis pigmentosa spinal muscular atrophyPrader-Willi syndrome Huntington diseasespinocerebellar ataxia myotonic dystrophyFragile-X syndrome  Or produce genetic susceptibility to disease: lupusbipolar disorder schizophreniamyocardial infarction type I diabetesasthma cardiac hypertrophymultiple sclerosis autoimmune diseaseselevated cholesterol