Building the preinitiation complex

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Chapter 22 Activating Transcription
Text assignment Lewin, Genes VIII pp. 631 to
652
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Domain structure of activators
Transcriptional activation motifs
DNA binding domain (DBD)
NLS
Make contact with basal factors and recruit them
to the promoter.
Dimerization domain
DNA
Upstream element
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Transcriptional activators.DNA binding domains
Zinc containing DNA binding domains
1. Zinc fingers Cys2-His2 coordinated by a
single zinc ion Zif268 2. Bimetal thiolate
cluster with 2 zinc ions coordinated by 6
cysteines GAL4 3. Nuclear receptors (steroids
related hormones) bind ligand to activate 2
zinc modules, (one for DNA interaction and the
other for dimerization) each with 4 cysteines.
Glucocorticoid receptor.
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Transcriptional activators
Zinc fingers
Zif268
Antiparallel b-strand and an a-helix
Three zinc fingers fit into the major groove
(Zif268)
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
5
Transcriptional activators
Zinc fingers
SP1 and Zif268
b-sheet
a-helix
Cys
His
Zn
Zn
Zn
Cys
His
Antiparallel b-strand and an a-helix
Three zinc fingers fit into the major groove
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
6
Transcriptional activators
GAL4 DNA binding domain is a bimetal thiolate
cluster
2 zinc ions 6 cysteines short a-helix
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
GAL4 dimer plus DNA
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GAL4 transactivator protein (DNA binding domain)
Leucine zipper for dimerization
2 zinc ions plus a short a-helix
2 zinc ions 6 cysteines a-helix
GAL4 dimer bound to DNA
bimetal thiolate cluster
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Leucine Zipper (coiled-coil)
Protein-protein interactions
Hydrophobic heptad repeat
abcdefg
leu
leu val ile ala
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Transcriptional activators
Nuclear receptors (steroid receptors)
2 zinc modules one binds DNA, the other
involved in dimerization each module contains 1
zinc ion and 4 cysteines
Must bind ligand (hormone) to activate transport
to the nucleus.
Glucocorticoid receptor
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
10
Transcriptional activators
DNA binding module
Glucocorticoid receptor dimer plus DNA
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
11
Steroid receptors
b-sheet
a-helix
cys or his
different
DNA binding
Cys
His
dimerization
Zn
Zn
Cys
His
Cys
His
DNA binding
Gly
Glu
Zn
Ser
Gly
Cys
His
Specificity of DNA recognition resides in aa
between the two His residues on the a-helix.
estrogen
glucocorticoid
Three zinc fingers fit into the major groove
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
12
Histone 1 DNA binding domain
Helix-turn-helix
This type of DBD is found in many transactivators
(HSFs homeodomain)
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Heat shock factor DNA binding domain
Helix-turn-helix
H
T
H
Triple helical cluster reinforced with b-strands
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Homeodomain proteins
Helix-turn-helix
Antennapedia phenotype resulting from a mutation
in the homeotic gene engrailed.
head
Homeodomain activators often control development
in organisms ranging from insects to plants.
legs
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
15
Basic leucine Zipper proteins (bZIP)
Leu zipper
Coiled-coil dimerization domain plus a basic
DNA interaction domain
basic
GCN4 yeast activator (requires SAGA complex)
GCN4 dimer plus DNA
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Basic Helix-loop-helix (bHLH)
Involved in differentiation of muscle cells.
From Molecular Biology (1999) by Robert Weaver,
McGraw Hill
MyoD transactivator
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Domain structure of activators
Transcriptional activation motifs
dimer
DNA binding domain (DBD)
NLS
Make contact with basal factors and recruit them
to the promoter.
Dimerization domain
DNA
Upstream element
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Acidic activation domains

• cluster of negative charges
• bulky hydrophobic residues
• (phe, ala, leu)

F
F
A
SSTP
amphipathic helix
amphipathic helix
Acidic activators are a major class and may be
the only one in yeast.
VP16 from the mammalian virus Herpes simplex is
the best characterized
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Synergism in TAF interactions with activators
Drosophila in vitro reactions Sauer, Hansen
Tjian (1995) Science 270 1783-1788 ? Synergism
only seen when BCD-Q and HB are both present with
TBP,TAF250, TAF60 and TAF110. 50- to 100-fold
stimulation compared to basal, versus 3- to
7-fold stimulation for a single activator/TAF
pair.
BCD bicoid domain
BCD-Q
HB hunchback
110
If the second transactivator contacts a different
target, then the stimulation is more than the sum
of either activator alone.
250
HB
60
TATAAA
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Synergism in TAF interactions with activators
Drosophila in vitro reactions ? in vitro
transcription reactions demonstrated activated
transcription using each of the isolated domains,
Q and A. Synergistic activation observed when
both domains are on the same protein, as in the
wt Bicoid. Reaction must contain TBP,TAF250,
TAF60 and TAF110.
Hd DNA binding
Q-rich
acidic
A-rich
Q
A
acidic
BCD-wt
-C
N-
Single activator causes synergistic activation if
it contains 2 different activation domains and
the promoter has multiple sites.
Note This assumes that the transactivator can
only contact one of its targets at a time, so
that there no competition for target binding by
the second transactivator.
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Synergism in TAF interactions with activators
Drosophila in vitro reactions Summary Two ways
to obtain synergism with TAF interactions
No competition for the same targets.
synergism
synergism
A
A
60
60
Q
250
250
110
110
Q
Q
TBP
TBP
A
1
2
1
2
Addition of a 2nd activator if each contain two
different activation motifs. Note synergistic
only if the transactivator can only contact a
single target at a time.
Addition of a 2nd activator if it contains a
single different activation motif.
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Additive TAF interactions with activators
Drosophila in vitro reactions
Competition for the same targets.
Additive effects
additive
additive
A
A
60
60
Q
250
250
110
110
Q
TBP
TBP
1
2
1
2
Addition of more of the same activator.
Addition of a 2nd activator that contains the
same activation motif.
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Additive TAF interactions with activators
Drosophila in vitro reactions
Competition for the same targets.
Additive effects
A
additive
additive
A
A
A
60
60
Q
250
250
110
110
Q
TBP
TBP
1
2
1
2
Addition of more of the same transactivator.
Additive since each AD competes for the same
target.
Addition of a 2nd transactivator that contains
the same activation motif.
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Modes of Activator Function
1. The Glucocorticord Receptor (GR)
glucocorticoid
DBD
N-
-C
Hormone binding
Required for activation
Glucocorticoid diffuses across the cellular
membrane and binds to the GR. This disrupts the
interaction between Hsp90 and the GR and
allows the hormone/receptor complex (HR) to
translocate to the nucleus where it dimerizes and
binds to the promoter to target genes
activating transcription.
If deleted, the GR is constitutive.
glucocorticoid
Hsp90
HR
GR

nucleus
Hsp90
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Modes of Activator Function
Ligand binding domain of the estrogen receptor
12
human
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Modes of Activator Function
Ligand binding domain of the glucocorticoid
receptor
P160 coactivator
A similar architecture is present in the
glucocorticoid receptor.
Helix 12
human
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P160 coactivator
Helix 12
taxmoxifen
Helix 12
estrogen
Taxmoxifen (used to fight breast cancer) causes
helix 12 to block p160 binding.
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Modes of Activator Function
Steroid Activators do not directly recruit
members of the preinitiation complex. Instead,
they make contact with coactivators, which in
turn, recruit basal factors.
Step 1 recruit HAT activity
Holo NZ
HR
HR
TBP
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Modes of Activator Function
The estrogen receptor first recruits the CBP in
the early stages of transcription, and later
recruits the Mediator.
Note CPB/p300 and PCAF leave as Mediator arrives.
Step 2 recruit Mediator
Holo NZ
HR
HR
TBP
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Estrogen receptor functions with coactivators
ligand binding domain
Estrogen receptor
Ligand binding causes Hsp90 to leave, which
unmasks region for p160 binding.
DNA binding domain
Estrogen receptor
Edwards, DE (2000) J. Mammary Gland Biol.
Neoplasma 5 307-324.
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P160 Nuclear receptor coactivators
1-p160 binds the ER 2- p300/CPB and CARM1 bind
p160
P300/CBP
CARM1
ER binding
HAT
p160
HAT
Basic HLH
Co-activator associated arginine methyltransferase
Edwards, DE (2000) J. Mammary Gland Biol.
Neoplasma 5 307-324.
Estrogen receptor
Note p160 has its own HAT activity and them
recruits other HAT and methyltransferase
activities.
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Modes of Activator Function
Other Steroid Activators
1. Glucocorticoid (GR), mineralcorticoid (MR),
androgen (AR), and progesterone (PR) all
form homodimers and recognize the sequence
TGTTCT. Half sites are palindromes. Spacing
determines type of element. The estrogen
receptor is the same, but its half site is
TGACCT. head to head receptors 2. Thyroid
(T3R), vitamin D (VDR), retinoic acid (RAR) and
9-cis-retinoic acid (RXR) receptors form
heterodimers recognizing half elements TGACCT
organized as direct repeats. Specific recognition
is influenced by spacing between repeats. 1
bp RXR (homodimer) 3 bp VDR 4 bp T3R 5 bp
RAR head to tail
RXR heterodimerizes with VDR, T3R and RAR.
glucocorticoid
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Modes of Activator Function
The thyroid receptor (TR) and the retinoic acid
receptor (RAR) bind the SMRT corepressor at the
promoter in the absence of ligand. Binding of
the ligand reduces affinity for the corepressor
and allows binding to coactivators.
SMRT corepressor
RXR
TR
TATAA
TR or RAR
coactivators
Holo NZ

CBP/p300
PCAF
hormone
HR
TBP