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Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 12
Transcription
Activators in
Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
12.1 Categories of Activators
• Activators can stimulate or inhibit
transcription by RNA polymerase II
• Structure is composed of at least 2
functional domains
– DNA-binding domain
– Transcription-activation domain
– Many also have a dimerization domain
12-2
DNA-Binding Domains
• Protein domain is an independently folded
region of a protein
• DNA-binding domains have DNA-binding
motif
– Part of the domain having characteristic
shape specialized for specific DNA binding
– Most DNA-binding motifs fall into 3 classes
12-3
Zinc-Containing Modules
• There are at least 3 kinds of zinccontaining modules that act as DNAbinding motifs
• All use one or more zinc ions to create a
shape to fit an a-helix of the motif into the
DNA major groove
– Zinc fingers
– Zinc modules
– Modules containing 2 zinc and 6 cysteines
12-4
Homeodomains
• These domains contain about 60 amino
acids
• Resemble the helix-turn-helix proteins in
structure and function
• Found in a variety of activators
• Originally identified in homeobox proteins
regulating fruit fly development
12-5
bZIP and bHLH Motifs
• A number of transcription factors have a
highly basic DNA-binding motif linked to
protein dimerization motifs
– Leucine zippers
– Helix-loop-helix
• Examples include:
– CCAAT/enhancer-binding protein
– MyoD protein
12-6
Transcription-Activating
Domains
• Most activators have one of these
domains
• Some have more than one
– Acidic domains such as yeast GAL4 with 11
acidic amino acids out of 49 amino acids in
the domain
– Glutamine-rich domains include Sp1 having 2
that are 25% glutamine
– Proline-rich domains such as CTF which has
a domain of 84 amino acids, 19 proline
12-7
12.2 Structures of the DNABinding Motifs of Activators
• DNA-binding domains have well-defined
structures
• X-ray crystallographic studies have shown
how these structures interact with their
DNA targets
• Interaction domains forming dimers, or
tetramers, have also been described
• Most classes of DNA-binding proteins
can’t bind DNA in monomer form
12-8
Zinc Fingers
• Described by Klug in TFIIIA
• Nine repeats of a 30-residue element:
– 2 closely spaced cysteines followed 12 amino
acids later by 2 closely spaced histidines
– Coordination of amino acids to the metal
helps form the finger-shaped structure
– Rich in zinc, enough for 1 zinc ion per repeat
– Specific recognition between the zinc finger
and its DNA target occurs in the major groove
12-9
Arrangement of Three Zinc
Fingers in a Curved Shape
The zinc finger is
composed of:
– An antiparallel b-strand
contains the 2 cysteines
– 2 histidines in an a-helix
– Helix and strand are
coordinated to a zinc ion
12-10
The GAL4 Protein
• The GAL4 protein is a member of the zinccontaining family of DNA-binding proteins
• It does not have a zinc finger
• Each GAL4 monomer contains a DNAbinding motif with:
– 6 cysteines that coordinate 2 zinc ions in a
bimetal thiolate cluster
– Short a-helix that protrudes into the DNA major
groove is the recognition module
– Dimerization motif with an a-helix that forms a
parallel coiled coil as it interacts with the a-helix
12-11
on another GAL4 monomer
The Nuclear Receptors
• A third class of zinc module is the nuclear
receptor
• This type of protein interacts with a variety
of endocrine-signaling molecules
• Protein plus endocrine molecule forms a
complex that functions as an activator by
binding to hormone response elements and
stimulating transcription of associated
genes
12-12
Type I Nuclear Receptors
• These receptors reside in the cytoplasm
bound to another protein
• When receptors bind to their hormone
ligands:
– Release their cytoplasmic protein partners
– Move to nucleus
– Bind to enhancers
– Act as activators
12-13
Glucocorticoid Receptors
• DNA-binding domain
with 2 zinc-containing
modules
• One module has most
DNA-binding residues
• Other module has the
surface for proteinprotein interaction to
form dimers
12-14
Types II and III Nuclear
Receptors
• Type II nuclear receptors stay within the
nucleus
• Bound to target DNA sites
• Without ligands the receptors repress gene
activity
• When receptors bind ligands, they activate
transcription
• Type III receptors are “orphan” whose
ligands are not yet identified
12-15
Homeodomains
• Homeodomains
contain DNA-binding
motif functioning as
helix-turn-helix motifs
• A recognition helix fits
into the DNA major
groove and makes
specific contacts there
• N-terminal arm nestles
in the adjacent minor
groove
12-16
The bZIP and bHLH Domains
• bZIP proteins dimerize through a leucine zipper
– This puts the adjacent basic regions of each
monomer in position to embrace DNA target like
a pair of tongs
• bHLH proteins dimerize through a helix-loophelix motif
– Allows basic parts of each long helix to grasp the
DNA target site
• bHLH and bHLH-ZIP domains bind to DNA in
the same way, later have extra dimerization
potential due to their leucine zippers
12-17
12.3 Independence of the
Domains of Activators
• DNA-binding and transcription-activating domains of
activator proteins are independent modules
• Making hybrid proteins with DNA-binding domain of one
protein, transcription-activating domain of another
• See that the hybrid protein still functions as an activator
12-18
12.4 Functions of Activators
• Bacterial core RNA polymerase is
incapable of initiating meaningful
transcription
• RNA polymerase holoenzyme can
catalyze basal level transcription
– Often insufficient at weak promoters
– Cells have activators to boost basal
transcription to higher level in a process
called recruitment
12-19
Eukaryotic Activators
• Eukaryotic activators also recruit RNA
polymerase to promoters
• Stimulate binding of general transcription
factors and RNA polymerase to a promoter
• 2 hypotheses for recruitment:
– General TF cause a stepwise build-up of
preinitiation complex
– General TF and other proteins are already
bound to polymerase in a complex called RNA
polymerase holoenzyme
12-20
Models for Recruitment
12-21
Recruitment of TFIID
• Acidic transcription-activating domain of
the herpes virus transcription factor VP16
binds to TFIID under affinity
chromatography conditions
• TFIID is rate-limiting for transcription in
some systems
• TFIID is the important target of the VP16
transcription-activating domain
12-22
Recruitment of the Holoenzyme
• Activation in some yeast promoters
appears to function by recruitment of
holoenzyme
• This is an alternative to the recruitment of
individual components of the holoenzyme
one at a time
• Some evidence suggests that recruitment
of the holoenzyme as a unit is not
common
12-23
Recruitment Model of GAL11Pcontaining Holoenzyme
• Dimerization domain
of FAL4 binds to
GAL11P in the
holoenzyme
• After dimerization, the
holoenzyme, along
with TFIID, binds to
the promoter,
activating the gene
12-24
12.5 Interaction Among
Activators
• General transcription factors must interact
to form the preinitiation complex
• Activators and general transcription factors
also interact
• Activators usually interact with one
another in activating a gene
– Individual factors interact to form a protein
dimer facilitating binding to a single DNA
target site
– Specific factors bound to different DNA target
sites can collaborate in activating a gene 12-25
Dimerization
• Dimerization is a great advantage to an
activator
• Dimerization increases the affinity
between activator and its DNA target
• Some activators form homodimers
• Heterodimers are also formed
– Products of the jun and fos genes form a
heterodimer
12-26
Action at a Distance
• Bacterial and eukaryotic enhancers
stimulate transcription even though located
some distance from their promoters
• Four hypotheses attempt to explain the
ability of enhancers to act at a distance
– Change in topology
– Sliding
– Looping
– Facilitated tracking
12-27
Hypotheses of Enhancer Action
12-28
Complex Enhancers
• Many genes can have more than one
activator-binding site permitting them to
respond to multiple stimuli
• Each of the activators that bind at these
sites must be able to interact with the
preinitiation complex assembling at the
promoter, likely by looping out any
intervening DNA
12-29
Control Region of the
Metallothionine Gene
• Gene product helps eukaryotes cope with heavy
metal poisoning
• Turned on by several different agents
12-30
Architectural Transcription
Factors
Architectural transcription factors are
those transcription factors whose sole or
main purpose seems to be to change the
shape of a DNA control region so that
other proteins can interact successfully to
stimulate transcription
12-31
An Architectural Transcription
Factor Example
• Within 112 bp
upstream of the start
of transcription are 3
enhancer elements
• These elements bind
to:
– Ets-1
– LEF-1
– CREB
12-32
Enhanceosome
• An enhanceosome is a
complex of enhancer
DNA with activators
contacting this DNA
• An example is the HMG
that helps to bend DNA
so that it may interact
with other proteins
12-33
DNA Bending Aids Protein
Binding
• The activator LEF-1 binds to the minor
groove of its DNA target through its HMG
domain and induces strong bending of
DNA
• LEF-1 does not enhance transcription by
itself
• Bending it induces helps other activators
bind and interact with activators and
general transcription factors
12-34
Examples of Architectural
Transcription Factors
• Besides LEF-1, HMG I(Y) plays a similar
role in the human interferon-b control gene
• For the IFN-b enhancer, activation seems
to require cooperative binding of several
activators, including HMG I(Y) to form an
enhanceosome with a specific shape
12-35
Insulators
Insulators act by:
• Enhancer-blocking
activity: insulator between
promoter and enhancer
prevents the promoter
from being activated
• Barrier activity: insulator
between promoter and
condensed, repressive
chromatin prevents
promoter from being
repressed
12-36
Mechanism of Insulator Activity
• Sliding model
– Activator bound to an
enhancer and stimulator
slides along DNA from
enhancer to promoter
• Looping model
– Two insulators flank an
enhancer, when bound
they interact with each
other isolating enhancer
12-37
Model of Multiple Insulator
Action
12-38
12.6 Regulation of Transcription
Factors
• Phosphorylation of activators can allow them to
interact with coactivators that in turn stimulate
transcription
• Ubiquitylation of transcription factors can mark
them for
– Destruction by proteolysis
– Stimulation of activity
• Sumoylation is the attachment of the polypeptide
SUMO which can target for incorporation into
compartments of the nucleus
• Methylation and acetylation can modulate activity
12-39
Phosphorylation and Activation
Replace this area with Figure 12.33:
A model for activation of a CRElinked gene
12-40
Activation of a Nuclear
Receptor-Activated Gene
12-41
Ubiquitylation
• Ubiquitylation, especially
monoubiquitylation, of some activators can
have an activating effect
• Polyubiquitylation marks these same
proteins for destruction
• Proteins from the 19S regulatory particle
of the proteasome can stimulate
transcription
12-42
Activator Sumoylation
• Sumoylation is the addition of one or more
copies of the 101-amino acid polypeptide
SUMO (Small Ubiquitin-Related Modifier)
to lysine residues on a protein
• Process is similar to ubiquitylation
• Results quite different – sumoylated
activators are targeted to a specific
nuclear compartment that keeps them
stable
12-43
Activator Acetylation
• Nonhistone activators and repressors can
be acetylated by HATs
• HAT is the enzyme histone
acetyltransferase which can act on
nonhistone activators and repressors
• Such acetylation can have either positive
or negative effects
12-44
Signal Transduction Pathways
• Signal transduction pathways begin with a
signaling molecule interacting with a
receptor on the cell surface
• This interaction sends the signal into the
cell and frequently leads to altered gene
expression
• Many signal transduction pathways rely on
protein phosphorylation to pass the signal
from one protein to another
• This leads to signal amplification at each
step
12-45
Three Signal Transduction
Pathways
12-46
Ras and Raf Signal Transduction
12-47
Wnt Signaling
12-48