Download Initiation of transcription by Pol II Separate basal and activated

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Presentation on genome editing
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
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
Transcription in Eukaryotes
Pol 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 synthesis
Cofactor = 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
+1
-30
TATAA
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
+1
UCE
-30
TATAA
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
Requires nucleosome repositioning
+1
UCE
-30
TATAA
coding sequence
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 II
Basal transcription
1) TFIID binds TATAA
box
2) 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 II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) TFIIH kinases CTD
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
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
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!
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])
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!
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
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
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!
Transcription
Template strand determines next base
Positioned by H-bonds
until RNA polymerase
links 5’ P to 3’ OH
in front
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
Transcription
NTP enters E site & rotates into A site
Transcription
NTP enters E site & rotates into A site
Specificity comes from trigger loop
Transcription
Specificity comes from trigger loop
Mobile motif that swings into position & triggers
catalysis
Transcription
Specificity comes from trigger loop
Mobile motif that swings into position & triggers
catalysis
Release of PPi
Triggers translocation
Transcription
Proofreading: when it makes a mistake it removes
~ 5 bases & tries again
Activated transcription by Pol II
Studied by mutating promoters for reporter genes
Activated transcription by Pol II
Studied by mutating promoters for reporter genes
Requires transcription factors and changes in chromatin
Activated transcription by Pol II
enhancers are sequences 5’ to TATAA
transcriptional activators bind them
• have distinct DNA binding and activation domains
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
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
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
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
Epigenetics
ChIP-chip & ChiP-seq data for whole genomes yield
complex 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 code
Methylation is more complex:
•H3K36me3 = on
•H3K27me3 = off
Histone code
Methylation is more complex:
•H3K36me3 = on
•H3K27me3 = off
•H3K4me1 = off
Histone code
Methylation is more complex:
•H3K36me3 = on
•H3K27me3 = off
•H3K4me1 = off
•H3K4me2 = primed
Histone code
Methylation is more complex:
•H3K36me3 = on
•H3K27me3 = off
•H3K4me1 = off
•H3K4me2 = primed
•H3K4me3 = on
Histone code
Modifications tend to group together: genes with
H3K4me3 also have H3K9ac
Histone code
Modifications tend to group together: genes with
H3K4me3 also have H3K9ac
Cytosine methylation is also associated with repressed
genes
Generating the histone code
Histone acetyltransferases add acetic acid
Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!
Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!
HATs are part of many complexes
Generating the histone code
Bromodomains specifically bind acetylated lysines
Generating the histone code
Bromodomains specifically bind acetylated lysines
Found in transcriptional activators & general TFs
Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate
Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate
Deacetylase mutants are sick!
Generating the histone code
CDK8 kinases histones to repress transcription
Generating the histone code
CDK8 kinases histones to repress transcription
Appears to interact with mediator to block transcription
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!
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
Generating the histone code
Rad6 proteins ubiquitinate histone H2B to repress
transcription
Generating the histone code
Rad6 proteins ubiquitinate histone H2B to repress
transcription
Polycomb proteins ubiquitinate histone H2A to silence
genes
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
Generating the histone code
Many proteins methylate histones: highly regulated!
Generating the histone code
Many proteins methylate histones: highly regulated!
Methylation status determines gene activity
Generating the histone code
Many proteins methylate histones: highly regulated!
Methylation status determines gene activity
Mutants (eg Curly leaf) are unhappy!
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)
Generating the histone code
Chromodomain protein HP1 can tell the difference
between H3K9me2 (yellow) & H3K9me3 (red)
Histone demethylases have been recently discovered
Generating methylated DNA
Si RNA are key: RNA Pol IV generates antisense or
foldback RNA, often from TE
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
Generating methylated DNA
RDR2 makes it DS, 24 nt siRNA are generated by DCL3
AGO4 binds siRNA, complex binds target & Pol V
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 AGO4siRNA to recruit “silencing Complex” to target site
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 AGO4siRNA to recruit “silencing Complex” to target site
Amplifies signal!
extends methylated region