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Regulating gene expression
Goal is
controlling
Proteins
•How many?
•Where?
•How active?
8 levels (two not
shown are mRNA
localization & prot
degradation)
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 gene models
• 20-40% of all RNA synthesis
• very sensitive to -aminitin
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) 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!
• even of genes that
aren’t expressed!
•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
•Recently identified “activating RNA”: bind enhancers &
mediator
Activated transcription by Pol II
•Other lncRNA “promote transcriptional poising” in
yeast
http://www.plosbiology.org/article/info%3Adoi%2F10.13
71%2Fjournal.pbio.1001715
•lncRNA displaces
glucose-responsive
repressors & corepressors from genes
for galactose catabolism
Activated transcription by Pol II
•Other lncRNA “promote transcriptional poising” in
yeast
http://www.plosbiology.org/article/info%3Adoi%2F10.13
71%2Fjournal.pbio.1001715
•lncRNA displaces
glucose-responsive
repressors & corepressors from genes
for galactose catabolism
•Speeds induction of
GAL genes
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
Generating methylated DNA
Si RNA are key: generated from antisense or
foldbackRNA
Generating methylated DNA
Si RNA are from antisense or foldback RNA
Primary 24 nt siRNA are generated by DCL3
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
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
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
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
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)
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
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation
Catalyzed by CEC attached to POLII
mRNA PROCESSING
1) Capping
2) Splicing: removal of introns
Evidence:
• electron microscopy
• sequence alignment
Splicing: the spliceosome cycle
1) U1 snRNP (RNA/protein complex) binds 5’ splice site
Splicing:The spliceosome cycle
1) U1 snRNP binds 5’ splice site
2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint
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
Splicing:
RNA is cut at 5’ splice site
cut end is trans-esterified to branchpoint A
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
Splicing:The spliceosome cycle
Splicing:
Some RNAs can self-splice!
role of snRNPs is to increase rate!
Why splice?
Splicing:
Why splice?
1) Generate diversity
exons often encode protein domains
Splicing:
Why splice?
1) Generate diversity
exons often encode protein domains
Introns = larger target for insertions,
recombination
Why splice?
1) Generate diversity
>94% of human genes show
alternate splicing
Why splice?
1) Generate diversity
>94% of human genes show
alternate splicing
same gene encodes
different protein
in different tissues
Why splice?
1) Generate diversity
>94% of human genes show
alternate splicing
same gene encodes
different protein
in different tissues
Stressed plants use
AS to make variant
stress-response
proteins
Why splice?
1) Generate diversity
>94% of human genes show
alternate splicing
same gene encodes
different protein
in different tissues
Stressed plants use
AS to make variant
Stress-response
proteins
Splice-regulator
proteins control AS:
regulated by cell-specific
expression and phosphorylation
Splicing:
Why splice?
1) Generate diversity
2) Modulate gene expression
introns affect amount of mRNA produced
mRNA Processing: RNA editing
Two types: C->U and A->I
mRNA Processing: RNA editing
Two types: C->U and A->I
• Plant mito and cp use C -> U
•>300 different editing events have been detected in plant
mitochondria: some create start & stop codons
mRNA Processing: RNA editing
Two types: C->U and A->I
• Plant mito and cp use C -> U
•>300 different editing events have been detected in plant
mitochondria: some create start & stop codons: way to
prevent nucleus from stealing genes!
mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a
stop codon @ aa 2153 (APOB48) cf full-length APOB100
• APOB48 lacks the CTD LDL receptor binding site
mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a
stop codon @ aa 2153 (APOB48) cf full-length APOB100
• APOB48 lacks the CTD LDL receptor binding site
• Liver makes APOB100 -> correlates with heart disease
mRNA Processing: RNA editing
Two types: C->U and A->I
• Adenosine de-aminases (ADA) are ubiquitously
expressed in mammals
• act on dsRNA & convert A to I (read as G)
mRNA Processing: RNA editing
Two types: C->U and A->I
• Adenosine de-aminases (ADA) are ubiquitously
expressed in mammals
• act on dsRNA & convert A to I (read as G)
• misregulation of A-to-I RNA editing has been implicated
in epilepsy, amyotrophic lateral sclerosis & depression
mRNA Processing: Polyadenylation
Addition of 200- 250 As to end of mRNA
Why bother?
• helps identify as mRNA
• required for translation
• way to measure age of mRNA
->mRNA s with < 200 As have short half-life
mRNA Processing: Polyadenylation
Addition of 200- 250 As to end of mRNA
Why bother?
• helps identify as mRNA
• required for translation
• way to measure age of mRNA
->mRNA s with < 200 As have short half-life
>50% of human mRNAs have alternative polyA sites!
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
• result : different mRNA, can result in altered export,
stability or different proteins
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
• result : different mRNA, can result in altered export,
stability or different proteins
• some thalassemias are due to mis-poly A
mRNA Processing: Polyadenylation
some thalassemias are due to mis-poly A
Influenza shuts down nuclear genes by preventing polyAdenylation (viral protein binds CPSF)
mRNA Processing: Polyadenylation
1) CPSF (Cleavage and Polyadenylation Specificity
Factor) binds AAUAAA in hnRNA
mRNA Processing: Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF (Cleavage Stimulatory Factor) binds G/U rich
sequence 50 bases downstream
CFI, CFII bind in between
Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF binds; CFI, CFII bind in between
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
mRNA Processing: Polyadenylation
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
4) PAP adds As slowly, CFI, CFII and CPSF fall off
mRNA Processing: Polyadenylation
4) PAP adds As slowly, CFI, CFII and CPSF fall off
5) PABII binds, add
As rapidly until 250
Coordination of mRNA processing
Splicing and polyadenylation factors bind CTD of RNA
Pol II-> mechanism to coordinate the three processes
Capping, Splicing and Polyadenylation
all start before transcription is done!
Export from Nucleus
Occurs through nuclear
pores
anything >
40 kDa needs
exportin
protein
bound
to 5’ cap
Export from Nucleus
In cytoplasm nuclear proteins fall off, new proteins bind
• eIF4E/eIF-4F bind cap
• also new
proteins bind
polyA tail
• mRNA is
ready to be
translated!