Regulating%20gene%20expression

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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)

2

• Transcription in Eukaryotes
• Pol I only makes 45S-rRNA precursor
• 50 of total RNA synthesis
• insensitive to ?-aminitin
• Mg2 cofactor
• Regulated _at_ initiation frequency

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RNA Polymerase III makes ribosomal 5S and tRNA
( some snRNA scRNA) gt100 different kinds of
genes 10 of all RNA synthesis Cofactor Mn2
cf Mg2 sensitive to high ?-aminitin
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• 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

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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
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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
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• 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
• arent expressed!
• Early elongation is also
• regulated!

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• Initiation of transcription by Pol II
• RNA pol II is paused on many promoters!
• even of genes that arent 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 arent expressed! (low
  mRNA)
• Early elongation is also
• regulated!
• PTEFb kinases CTD to
• stimulate processivity
• processing
• Many genes have
• short transcripts

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• Initiation of transcription by Pol II
• RNA pol II is paused on many promoters!
• even of genes that arent 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!

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Transcription Template strand determines next
base Positioned by H-bonds until RNA
polymerase links 5 P to 3 OH in front
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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
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Transcription NTP enters E site rotates into A
site
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Transcription NTP enters E site rotates into A
site Specificity comes from trigger loop
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Transcription Specificity comes from trigger
loop Mobile motif that swings into position
triggers catalysis
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Transcription Specificity comes from trigger
loop Mobile motif that swings into position
triggers catalysis Release of PPi triggers
translocation
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Transcription Proofreading when it makes a
mistake it removes 5 bases tries again
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Activated transcription by Pol II Studied by
mutating promoters for reporter genes
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Activated transcription by Pol II Studied by
mutating promoters for reporter genes Requires
transcription factors and changes in chromatin
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• Activated transcription by Pol II
• enhancers are sequences 5 to TATAA
• transcriptional activators bind them
• have distinct DNA binding and activation domains

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• 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

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• 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

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• Activated transcription by Pol II
• Other lncRNA promote transcriptional poising in
  yeast
• http//www.plosbiology.org/article/info3Adoi2F10
  .13712Fjournal.pbio.1001715
• lncRNA displaces
• glucose-responsive
• repressors co-
• repressors from genes
• for galactose catabolism

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• Activated transcription by Pol II
• Other lncRNA promote transcriptional poising in
  yeast
• http//www.plosbiology.org/article/info3Adoi2F10
  .13712Fjournal.pbio.1001715
• lncRNA displaces
• glucose-responsive
• repressors co-
• repressors from genes
• for galactose catabolism
• Speeds induction of
• GAL genes

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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
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• First remodel chromatin
• some proteins reposition
• nucleosomes
• others acetylate histones
• Neutralizes ve charge
• makes them release DNA

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

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Epigenetics ChIP-chip ChiP-seq data for whole
genomes yield complex picture 17 mods are
associated with active genes in CD-4 T cells
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• Generating methylated DNA
• Si RNA are key generated from antisense or
  foldbackRNA

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

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• 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

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• 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

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• 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!-gt extends
• Methylated region

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• 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!-gt extends
• Methylated region
• These guide silencing
• Complex to target site
• (includes Cytosine H3K9
• Methyltransferases)

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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
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mRNA PROCESSING Primary transcript is
hnRNA undergoes 3 processing reactions before
export to cytosol 1) Capping addition of 7-methyl
G to 5 end
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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
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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
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• mRNA PROCESSING
• 1) Capping
• 2) Splicing removal of introns
• Evidence
• electron microscopy
• sequence alignment

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Splicing the spliceosome cycle 1) U1 snRNP
(RNA/protein complex) binds 5 splice site
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SplicingThe spliceosome cycle 1) U1 snRNP binds
5 splice site 2) U2 snRNP binds
branchpoint -gt displaces A at branchpoint
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SplicingThe spliceosome cycle 1) U1 snRNP binds
5 splice site 2) U2 snRNP binds
branchpoint -gt displaces A at branchpoint 3)
U4/U5/U6 complex binds intron displace
U1 spliceosome has now assembled
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Splicing RNA is cut at 5 splice site cut end
is trans-esterified to branchpoint A
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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 -gt lariat intron is
degraded
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SplicingThe spliceosome cycle
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Splicing Some RNAs can self-splice! role of
snRNPs is to increase rate! Why splice?
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Splicing Why splice? 1) Generate diversity
exons often encode protein domains
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Splicing Why splice? 1) Generate
diversity exons often encode protein
domains Introns larger target for insertions,
recombination
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Why splice? 1) Generate diversity gt94 of human
genes show alternate splicing
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Why splice? 1) Generate diversity gt94 of human
genes show alternate splicing same gene encodes
different protein in different tissues
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Why splice? 1) Generate diversity gt94 of human
genes show alternate splicing same gene encodes
different protein in different tissues Stressed
plants use AS to make variant stress-response
proteins
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Why splice? 1) Generate diversity gt94 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
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Splicing Why splice? 1) Generate diversity 2)
Modulate gene expression introns affect amount of
mRNA produced
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mRNA Processing RNA editing Two types C-gtU and
A-gtI
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• mRNA Processing RNA editing
• Two types C-gtU and A-gtI
• Plant mito and cp use C -gt U
• gt300 different editing events have been detected
  in plant mitochondria some create start stop
  codons

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• mRNA Processing RNA editing
• Two types C-gtU and A-gtI
• Plant mito and cp use C -gt U
• gt300 different editing events have been detected
  in plant mitochondria some create start stop
  codons way to prevent nucleus from stealing
  genes!

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• mRNA Processing RNA editing
• Human intestines edit APOB mRNA C -gt U to create
  a stop codon _at_ aa 2153 (APOB48) cf full-length
  APOB100
• APOB48 lacks the CTD LDL receptor binding site

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• mRNA Processing RNA editing
• Human intestines edit APOB mRNA C -gt U to create
  a stop codon _at_ aa 2153 (APOB48) cf full-length
  APOB100
• APOB48 lacks the CTD LDL receptor binding site
• Liver makes APOB100 -gt correlates with heart
  disease

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• mRNA Processing RNA editing
• Two types C-gtU and A-gtI
• Adenosine de-aminases (ADA) are ubiquitously
  expressed in mammals
• act on dsRNA convert A to I (read as G)

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• mRNA Processing RNA editing
• Two types C-gtU and A-gtI
• 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

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• 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
• -gtmRNA s with lt 200 As have short half-life

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• 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
• -gtmRNA s with lt 200 As have short half-life
• gt50 of human mRNAs have alternative polyA sites!

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mRNA Processing Polyadenylation gt50 of human
mRNAs have alternative polyA sites!
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• mRNA Processing Polyadenylation
• gt50 of human mRNAs have alternative polyA sites!
• result different mRNA, can result in altered
  export, stability or different proteins

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• mRNA Processing Polyadenylation
• gt50 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

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mRNA Processing Polyadenylation some
thalassemias are due to mis-poly A Influenza
shuts down nuclear genes by preventing
poly-Adenylation (viral protein binds CPSF)
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mRNA Processing Polyadenylation 1) CPSF
(Cleavage and Polyadenylation Specificity Factor)
binds AAUAAA in hnRNA
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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
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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
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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
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• mRNA Processing Polyadenylation
• 4) PAP adds As slowly, CFI, CFII and CPSF fall
  off
• PABII binds, add
• As rapidly until 250

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Coordination of mRNA processing Splicing and
polyadenylation factors bind CTD of RNA Pol II-gt
mechanism to coordinate the three
processes Capping, Splicing and Polyadenylation
all start before transcription is done!
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Export from Nucleus Occurs through nuclear
pores anything gt 40 kDa needs
exportin protein bound to 5 cap
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• 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!