Regulating gene expression Goal is controlling Proteins How many? Where? How active? - PowerPoint PPT Presentation

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Regulating gene expression Goal is controlling Proteins How many? Where? How active?
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Regulating gene expression Goal is controlling Proteins How many? Where? How active?

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  1. Regulating gene expression • Goal is • controlling • Proteins • How many? • Where? • How active? • 8 levels (two not • shown are mRNA • localization & prot • degradation)

  2. Transcription in Eukaryotes • Pol I: only makes 45S-rRNA precursor • 50 % of total RNA synthesis • insensitive to-aminitin • Mg2+ cofactor • Regulated @ initiation frequency

  3. RNA Polymerase III makesribosomal 5SandtRNA (+ some snRNA & scRNA) >100 different kinds of genes ~10% of all RNA synthesis Cofactor = Mn2+ cfMg2+ sensitive to high [-aminitin]

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

  5. 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

  6. 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

  7. 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! • Early elongation is also • regulated!

  8. 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

  9. 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

  10. 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!

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

  12. 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

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

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

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

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

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

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

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

  20. Activated transcription by Pol II • enhancers are sequences 5’ to TATAA • transcriptional activators bind them • have distinct DNA bindingandactivation domains

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

  22. Activated transcription by Pol II • enhancers are sequences 5’ to TATAA • transcriptional activators bind them • have distinct DNA bindingandactivation domains • activation domain interacts with mediator • helps assemble initiation complex on TATAA • Recently identified “activating RNA”: bind enhancers & mediator

  23. Activated transcription by Pol II • Other lncRNA “promote transcriptional poising” in yeast • http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001715 • lncRNA displaces • glucose-responsive • repressors & co- • repressors from genes • for galactose catabolism

  24. Activated transcription by Pol II • Other lncRNA “promote transcriptional poising” in yeast • http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001715 • lncRNA displaces • glucose-responsive • repressors & co- • repressors from genes • for galactose catabolism • Speeds induction of • GAL genes

  25. Euk gene regulation Initiating transcription is 1st & 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

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

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

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

  29. Generating methylated DNA • Si RNA are key: generated from antisense or foldbackRNA

  30. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3

  31. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA

  32. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA • RDR2 makes bottom strand

  33. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA • RDR2 makes bottom strand • DCL3 cuts dsRNA into 24nt • 2˚ siRNA

  34. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA • RDR2 makes bottom strand • DCL3 cuts dsRNA into 24nt • 2˚ siRNA • Amplifies signal!-> extends • Methylated region

  35. Generating methylated DNA • Si RNA are from antisense or foldback RNA • Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA • RDR2 makes bottom strand • DCL3 cuts dsRNA into 24nt • 2˚ siRNA • Amplifies signal!-> extends • Methylated region • These guide “silencing • Complex” to target site • (includes Cytosine & H3K9 • Methyltransferases)

  36. mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

  37. mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1)Capping addition of 7-methyl G to 5’ end

  38. mRNA PROCESSING • Primary transcript is hnRNA • undergoes 3 processing reactions before export to cytosol • 1)Cappingaddition of 7-methyl G to 5’ end • identifies it as mRNA: needed for export & translation

  39. mRNA PROCESSING • Primary transcript is hnRNA • undergoes 3 processing reactions before export to cytosol • 1)Cappingaddition of 7-methyl G to 5’ end • identifies it as mRNA: needed for export & translation • Catalyzed by CEC attached to POLII

  40. mRNA PROCESSING • 1) Capping • 2) Splicing: removal of introns • Evidence: • electron microscopy • sequence alignment

  41. Splicing: the spliceosome cycle 1)U1 snRNP (RNA/protein complex) binds 5’ splice site

  42. Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNPbinds “branchpoint” -> displaces A at branchpoint

  43. Splicing:The spliceosome cycle • 1) U1 snRNP binds 5’ splice site • 2) U2 snRNP binds “branchpoint” • -> displaces A at branchpoint • 3) U4/U5/U6 complex • binds intron • displace U1 • spliceosome • has now assembled

  44. Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpointA

  45. Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded

  46. Splicing:The spliceosome cycle

  47. Splicing: • Some RNAs can self-splice! • role of snRNPs is to increase rate! • Why splice?

  48. Splicing: • Why splice? • 1) Generate diversity • exons often encode protein domains

  49. Splicing: • Why splice? • 1) Generate diversity • exons often encode protein domains • Introns = larger target for insertions, recombination

  50. Why splice? 1) Generate diversity >94% of human genes show alternate splicing