Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 12
Transcription Activators
in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transcription Activators of Eukaryotes
• The general transcription factors by themselves
dictate the starting point and direction of
transcription but they are capable of sponsoring
only a low level of transcription or basal
transcription
• Transcription of active genes in cells rises above
the basal level
• Eukaryotic cells have additional, gene-specific
transcription factors called activators that bind
to DNA elements called enhancers to provide
the extra needed boost to transcription
12-2
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
12-3
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;
zinc-containing modules, homeodomains and
bZIP and bHLH motifs
12-4
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
12-5
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
12-6
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
12-7
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
12-8
Summary
• Eukaryotic activators are composed of at
least two domains: a DNA-binding domain
and a transcription-activating domain
• DNA-binding domains contain motifs such
as zinc modules, homeodomains, and
bZIP or bHLH motifs
• Transcription activating domains can be
acidic, glutamine-rich or proline-rich
12-9
12.2 Structures of the DNA-Binding 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
12-10
Zinc Fingers
• Described by Klug in GTF 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
12-11
Arrangement of Three Zinc Fingers in a
Curved Shape
The zinc finger is
composed of:
– An antiparallel b-strand
that contains 2 cysteines
– 2 histidines in an a-helix
– Helix and strand are
coordinated to a zinc ion
12-12
The GAL4 Protein
• The GAL4 protein is a member of the zinccontaining family of DNA-binding proteins
• 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
on another GAL4 monomer
12-13
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
12-14
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
12-15
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
12-16
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
12-17
Homeodomain-DNA Complex
• Homeodomains contain
DNA-binding motif
functioning as helix-turnhelix motifs
• A recognition helix fits
into the DNA major
groove and makes
specific contacts there
• N-terminal arm nestles in
the adjacent minor
groove
12-18
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
12-19
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
• The hybrid protein still functions as an activator
12-20
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
12-21
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
12-22
Models for Recruitment of Preinitiation
Complex Components in Yeast
12-23
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
12-24
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
12-25
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
12-26
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
12-27
Dimerization
• Dimerization is a great advantage to an
activator as it increases the affinity
between the activator and its DNA target
• Some activators form homodimers but
others function as heterodimers
12-28
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
12-29
Hypotheses of Enhancer Action
12-30
3C: Method to detect DNA looping
• Chromosome conformation capture (3C) is
a technique used to determine if enhancer
action requires DNA looping
• Used to test whether two remote DNA
regions, such as an enhancer and a
promoter, are brought together
12-31
Genomic Imprinting
• Because most eukaryotes are diploid organisms,
you would predict that it does not matter which
allele of any given gene came form the mother
or the father
• This is true in most cases but there are
important exceptions
• The differences between the genes resides in
how they are modified, or imprinted, differently in
females and males
• Evidence exists in mice and humans
12-32
Transcription Factories
• Discrete nuclear sites where transcription
of multiple genes occurs
• If two or more genes on the same
chromosome are clustered in the same
transcription factory, DNA loops would
naturally form between them
• Thus, the existence of transcription
factories implies the existence of DNA
loops in eukaryotic cells
12-33
Evidence for Transcription Factories
• Cook and colleagues counted the number of
transcription factories by labeling growing RNA
chains in HeLa cells with BrU followed by
permeabilization and further labeling with biotinCTP detected with anti-BrU or anti-biotin primary
antibodies followed by secondary antibodies
labeled with gold particles
• They concluded that transcription is associated
with the clusters, not the single particles
12-34
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
12-35
Control Region of the Metallothionine Gene
• The metallothionine gene product helps
eukaryotes cope with heavy metal poisoning
• Turned on by several different agents
• Complex enhancers enable a gene to respond
differently to different combinations of activators
• This gives cells exquisitely fine control over their
genes in different tissues, or at different times in
a developing organism
12-36
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
12-37
Example of Architectural Transcription
Factor: Control region of the human TCR a chain gene
• Within 112 bp upstream of the start of
transcription are 3 enhancer elements
• These elements bind to:
– Ets-1, LEF-1, CREB
12-38
Enhanceosome
• An enhanceosome is a nucleoprotein complex
containing a collection of activators bound to
an enhancer in such a way that stimulates
transcription
• The archetypal enhanceosome involves the
IFNb enhancer with a structure that involves
eight polypeptides bound cooperatively to an
essentially straight 55-bp strech of DNA
12-39
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
12-40
Insulators
• Insulators can shield genes from activation by
enhancers (enhancer blocking activity)
• Insulators can shield genes from repression by
silencers (barrier activity)
12-41
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
12-42
Model of Multiple Insulator Action
12-43
Summary
• Some insulators have both enhancer-blocking and
barrier activities, but some have only one or the
other
• Insulators may do their job by working in pairs that
bind proteins that can interact to form DNA loops
that would isolate enhancers and silencers so they
can no longer stimulate or repress promoters
• Insulators may establish boundaries between DNA
regions in a chromosome
12-44
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
12-45
Phosphorylation and Activation: A Model
for activation of a CRE-linked gene
Replace this area with Figure 12.33:
A model for activation of a CRElinked gene
12-46
Model for the Activation of a Nuclear
Receptor-Activated Gene
12-47
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
12-48
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
12-49
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
12-50
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
12-51
Three pathways that use CBP/p300 to
mediate transcription activation
12-52
Ras and Raf Signal Transduction
12-53