next assignment presentation on genome editing
TRANSCRIPT
Next AssignmentPresentation on genome editing http://www.sciencemag.org/content/339/6121/768.full1.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
Transcription in Eukaryotes3 RNA polymerasesall are multi-subunit complexes 5 in common 3 very similar variable # unique onesNow have Pols IV & V in plantsMake siRNA
Transcription in EukaryotesPol I: only makes 45S-rRNA precursor
• 50 % of total RNA synthesis• insensitive to -aminitin•Mg2+ cofactor•Regulated @ initiation frequency
RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA & scRNA)>100 different kinds of genes ~10% of all RNA synthesisCofactor = Mn2+ cf Mg2+
sensitive to high [-aminitin]
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
RNA Polymerase II
12 subunits in yeast,
unknown elsewhere
Largest subunit (L’) has
CarboxyTerminal
Domain (CTD)
important role in
regulating pol II
Initiation of transcription by Pol II
Needs > 30 other factors to initiate transcription
final complex is called a transcriptosome
contains > 50 proteins
Initiation of transcription by Pol II
Separate basal and activated transcription
basal transcription is not regulated
driven by minimal promoter
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
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 sequenceUCE
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 sequenceUCE
Initiation of transcription
by Pol II
Basal transcription
1) TFIID binds to
TATAA box
2) Distorts DNA
Initiation of transcription by Pol IIBasal transcription1) TFIID binds TATAA box2) TFIIA and TFIIB bind TFIID/DNA
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
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
Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) TFIIH kinases CTD
Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) TFIIH kinases CTD
negative charge gets it started
3) Exchange initiation for elongation factors
Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) Kinase CTD
negative charge gets it started
3) Exchange initiation for elongation factors4) Continues untilhits terminator
Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) Kinase CTD
negative charge gets it started
3) RNA pol II is pausedon many promoters!
Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) Kinase CTD
negative charge gets it started
3) RNA pol II is pausedon many promoters!• even of genes thataren’t expressed!(low [mRNA])
Initiation of transcription by Pol IIRNA pol II is paused on many promoters!• even of genes that aren’t expressed! (low [mRNA])•Early elongation is also regulated!
Initiation of transcription by Pol IIRNA 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
Initiation of transcription by Pol IIRNA 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
Initiation of transcription by Pol IIRNA 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!
TranscriptionTemplate strand determines next basePositioned by H-bondsuntil RNA polymeraselinks 5’ P to 3’ OH in front
TranscriptionTemplate strand determines next basePositioned by H-bondsuntil RNA polymeraselinks 5’ P to 3’ OH in frontEnergy comes from hydrolysisof 2 Pi
TranscriptionSpecificity comes from trigger loopMobile motif that swings into position & triggers catalysis
TranscriptionSpecificity comes from trigger loopMobile motif that swings into position & triggers catalysisRelease of PPi Triggers translocation
Activated transcription by Pol IIStudied by mutating promoters for reporter genesRequires transcription factors and changes in chromatin
Activated transcription by Pol IIenhancers are sequences 5’ to TATAA
transcriptional activators bind them• have distinct DNA binding and activation domains
Activated transcription by Pol IIenhancers 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
Activated transcription by Pol IIenhancers 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
Euk gene regulationInitiating transcription is 1st & most important controlMost genes are condensedonly express needed genesnot enough room in nucleus toaccess all genes at same time!must find & decompress gene
First “remodel” chromatin:• some proteins reposition nucleosomes • others acetylate histones• Neutralizes +ve charge• makes them release DNA
EpigeneticsChIP-chip & ChiP-seq data for whole genomes yieldcomplex picture: 17 mods are associated with active genes in CD-4 T cells
Epigenetics• various chromatin modifications are associated with activated & repressed genes•Acetylation, egH3K9Ac, is associated with active genes
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
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!
Histone code•Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation shows condensation• Ubiquitination of H2A and H2B shows repression
Histone code•Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation shows condensation• Ubiquitination of H2A and H2B shows repression• also marks DNA damage
Histone code•Acetylation, egH3K9Ac, is associated with active genes• Phosphorylation shows condensation• Ubiquitination of H2A and H2B shows repression• Methylation is more complex:
Histone codeMethylation is more complex:•H3K36me3 = on•H3K27me3 = off•H3K4me1 = off•H3K4me2 = primed
Histone codeMethylation is more complex:•H3K36me3 = on•H3K27me3 = off•H3K4me1 = off•H3K4me2 = primed•H3K4me3 = on
Histone codeModifications tend to group together: genes with H3K4me3 also have H3K9acCytosine methylation is also associated with repressed genes
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!HATs are part of many complexes
Generating the histone codeBromodomains specifically bind acetylated lysinesFound in transcriptional activators & general TFs
Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcription
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!
Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!Phosphatases reset the genes
Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genes
Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genesA TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing
Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activity
Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!
Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow)& H3K9me3 (red)
Generating the histone codeChromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)Histone demethylases have been recently discovered
Generating methylated DNASi RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE
Generating methylated DNASi RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TERDR2 makes it DS, 24 nt siRNA are generated by DCL3
Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol V
Generating methylated DNARDR2 makes it DS, 24 nt siRNA are generated by DCL3AGO4 binds siRNA, complex binds target & Pol VPol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site