Next Assignment Presentation on genome editing .

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

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 (includingTBP) binds TATAA box

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

TranscriptionNTP enters E site & rotates into A site

TranscriptionNTP enters E site & rotates into A siteSpecificity comes from trigger loop

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

TranscriptionProofreading: when it makes a mistake it removes ~ 5 bases & tries again

Activated transcription by Pol IIStudied by mutating promoters for reporter genes

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

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

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

Histone codeMethylation is more complex:•H3K36me3 = on•H3K27me3 = off•H3K4me1 = off

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 H3K9ac

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 acid

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 lysines

Generating the histone codeBromodomains specifically bind acetylated lysinesFound in transcriptional activators & general TFs

Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetate

Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!

Generating the histone codeCDK8 kinases histones to repress transcription

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 transcription

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!

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

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 siteAmplifies signal!extends meth-ylated region