1. Next Assignment Presentation on genome editing http://www.sciencemag.org/content/339/6121/768.full http://www.sciencemag.org/content/339/6121/768.full 1.Crispr- associated nucleases 2.Homing endonucleases 3.Zinc-finger nucleases 4.Transcription activator-like effector nucleases (TALENs) 5.Triple-helix–forming oligonucleotides conjugated to restriction endonucleases 6.CRE/LOX system

2. Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Now have Pols IV & V in plants Make siRNA

3. Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to  -aminitin Mg 2+ cofactor Regulated @ initiation frequency

4. RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA & scRNA) >100 different kinds of genes ~10% of all RNA synthesis Cofactor = Mn 2+ cf Mg 2+ sensitive to high [  -aminitin]

5. RNA Polymerase II makes mRNA (actually hnRNA), some snRNA and scRNA ~ 30,000 different genes 20-40% of all RNA synthesis very sensitive to  -aminitin

6. RNA Polymerase II 12 subunits in yeast, unknown elsewhere

7. RNA Polymerase II 12 subunits in yeast, unknown elsewhere Largest subunit (L’) has CarboxyTerminal Domain (CTD) important role in regulating pol II

8. Initiation of transcription by Pol II Needs > 30 other factors to initiate transcription final complex is called a transcriptosome contains > 50 proteins

9. Initiation of transcription by Pol II Separate basal and activated transcription basal transcription is not regulated driven by minimal promoter

10. Initiation of transcription by Pol II Separate basal and activated transcription basal transcription is not regulated driven by minimal promoter TATAA box at -30 TATAA -30 +1 coding sequence

11. Initiation of transcription by Pol II Separate basal and activated transcription activated transcription is regulated by proteins bound to promoter elements called enhancers and silencers usually 5’ to TATAA box TATAA -30 +1 coding sequence UCE

12. Initiation of transcription by Pol II Separate basal and activated transcription activated transcription is regulated by proteins bound to promoter elements called enhancers and silencers usually 5’ to TATAA box Requires nucleosome repositioning TATAA -30 +1 coding sequence UCE

14. Initiation of transcription by Pol II Basal transcription 1) TFIID (includingTBP) binds TATAA box

15. Initiation of transcription by Pol II Basal transcription 1) TFIID binds to TATAA box 2) Distorts DNA

16. Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind TFIID/DNA

17. Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind TFIID/DNA 3) Complex recruits Pol II

18. Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind to TFIID/DNA 3) Complex recruits Pol II 4) Still must recruit TFIIE & TFIIH to form initiation complex

19. Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) TFIIH kinases CTD

20. Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) TFIIH kinases CTD negative charge gets it started 3) Exchange initiation for elongation factors

21. Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) Exchange initiation for elongation factors 4) Continues until hits terminator

22. Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) RNA pol II is paused on many promoters!

23. Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA])

24. Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated!

25. Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing

26. Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing Many genes have short transcripts

27. Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing Many genes have short transcripts Yet another new level of control!

28. Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front

29. Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front Energy comes from hydrolysis of 2 Pi

30. Transcription NTP enters E site & rotates into A site

31. Transcription NTP enters E site & rotates into A site Specificity comes from trigger loop

32. Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis

33. Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis Release of PPi Triggers translocation

34. Transcription Proofreading: when it makes a mistake it removes ~ 5 bases & tries again

35. Activated transcription by Pol II Studied by mutating promoters for reporter genes

36. Activated transcription by Pol II Studied by mutating promoters for reporter genes Requires transcription factors and changes in chromatin

37. Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains

38. Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains activation domain interacts with mediator helps assemble initiation complex on TATAA

39. Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains activation domain interacts with mediator helps assemble initiation complex on TATAA

40. Euk gene regulation Initiating transcription is 1 st & most important control Most genes are condensed only express needed genes not enough room in nucleus to access all genes at same time! must find & decompress gene

41. First “remodel” chromatin: some proteins reposition nucleosomes others acetylate histones Neutralizes +ve charge makes them release DNA

42. Epigenetics heritable chromatin modifications are associated with activated & repressed genes

43. Epigenetics ChIP-chip & ChiP-seq data for whole genomes yield complex picture: 17 mods are associated with active genes in CD-4 T cells

44. Epigenetics various chromatin modifications are associated with activated & repressed genes Acetylation, egH3K9Ac, is associated with active genes

45. Epigenetics various chromatin modifications are associated with activated & repressed genes Acetylation, egH3K9Ac, is associated with active genes Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation

46. Epigenetics various chromatin modifications are associated with activated & repressed genes Acetylation, egH3K9Ac, is associated with active genes Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation but, H3S10 + H3K14ac = active!

47. Histone code Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression

48. Histone code Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression also marks DNA damage

49. Histone code Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression Methylation is more complex:

50. Histone code Methylation is more complex: H3K36me3 = on H3K27me3 = off

51. Histone code Methylation is more complex: H3K36me3 = on H3K27me3 = off H3K4me1 = off

52. Histone code Methylation is more complex: H3K36me3 = on H3K27me3 = off H3K4me1 = off H3K4me2 = primed

53. Histone code Methylation is more complex: H3K36me3 = on H3K27me3 = off H3K4me1 = off H3K4me2 = primed H3K4me3 = on

54. Histone code Modifications tend to group together: genes with H3K4me3 also have H3K9ac

55. Histone code Modifications tend to group together: genes with H3K4me3 also have H3K9ac Cytosine methylation is also associated with repressed genes

56. Generating the histone code Histone acetyltransferases add acetic acid

57. Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick!

58. Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick! HATs are part of many complexes

59. Generating the histone code Bromodomains specifically bind acetylated lysines

60. Generating the histone code Bromodomains specifically bind acetylated lysines Found in transcriptional activators & general TFs

61. Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate

62. Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate Deacetylase mutants are sick!

63. Generating the histone code CDK8 kinases histones to repress transcription

64. Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription

65. Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes!

66. Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes! Phosphatases reset the genes

67. Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription

68. Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes

69. Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing

70. Generating the histone code Many proteins methylate histones: highly regulated!

71. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity

72. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy!

73. Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy! Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)

74. Generating the histone code Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red) Histone demethylases have been recently discovered

75. Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE

76. Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE RDR2 makes it DS, 24 nt siRNA are generated by DCL3

77. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V

78. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4- siRNA to recruit “silencing Complex” to target site

79. Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4- siRNA to recruit “silencing Complex” to target site Amplifies signal! extends meth- ylated region