Chapter 15
*Lecture Outline
*See separate FlexArt PowerPoint slides for all
figures and tables pre-inserted into PowerPoint
without notes.
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INTRODUCTION


Eukaryotic organisms derive many benefits from
regulating their genes
For example



They can respond to changes in nutrient availability
They can respond to environmental stresses
In plants and animals, multicellularity and a more
complex cell structure also demand a much greater
level of gene regulation
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15-2
INTRODUCTION

Gene regulation is necessary to ensure

1. Expression of genes in an accurate pattern during the
various developmental stages of the life cycle


2. Differences among distinct cell types


Some genes are only expressed during embryonic stages,
whereas others are only expressed in the adult
Nerve and muscle cells look so different because of gene
regulation rather than differences in DNA content
Figure 15.1 describes the steps of gene expression
that are regulated in eukaryotes
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15-3
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REGULATION OF
GENE EXPRESSION
Gene
Transcription
Regulatory transcription factors
activate or inhibit transcription.
The arrangements and composition
of nucleosomes influence transcription.
DNA methylation (usually) inhibits
transcription.
pre-mRNA
Alternative splicing alters exon
RNA
choices.
processing
RNA editing alters the base
sequence of mRNAs.
mRNA
Translation
Small RNAs, called miRNAs and siRNAs, silence the
translation of mRNA.
Phosphorylation of translational initiation factors
may regulate translation.
Proteins that bind to the 5′ end of mRNA regulate translation.
mRNA stability may be influenced by RNA binding proteins.
Protein
Posttranslational
Feedback inhibition and covalent modifications
modifications
regulate protein function.
Functional protein
Figure 15.1
15-4
15.1 REGULATORY
TRANSCRIPTION FACTORS


Transcription factors are proteins that influence the
ability of RNA polymerase to transcribe a given gene
There are two main types

General transcription factors



Required for the binding of the RNA pol to the core promoter and
its progression to the elongation stage
Are necessary for basal transcription
Regulatory transcription factors


Serve to regulate the rate of transcription of target genes
They influence the ability of RNA pol to begin transcription of a
particular gene
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15-5

Regulatory transcription factors recognize cis
regulatory elements located near the core promoter


These sequences are known as control elements or
regulatory elements
The binding of regulatory transcription factors to
control elements affects the transcription of an
associated gene

A regulatory protein that increases the rate of
transcription is termed an activator


A regulatory protein that decreases the rate of
transcription is termed a repressor


The sequence it binds is called an enhancer
The sequence it binds is called a silencer
Refer to Figure 15.2
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15-6
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RNA polymerase
and general
transcription
factors
Activator
protein
Enhancer
Core
promoter
RNA transcription
is increased.
(a) Gene activation
Repressor
protein
Silencer
Core
promoter
RNA transcription
is inhibited.
(b) Gene repression
Figure 15.2
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15-7

Most Eukaryotic genes are regulated by many factors


This is known as combinatorial control
Common factors contributing to combinatorial control are:



One or more activator proteins may stimulate transcription
One or more repressor proteins may inhibit transcription
Activators and repressors may be modulated by:





Regulatory proteins may alter nucleosomes near the promoter
DNA methylation may inhibit transcription



binding of small effector molecules
protein-protein interactions
covalent modifications
prevent binding of an activator protein
recruiting proteins that compact the chromatin
Various combinations of these factors can contribute to the
regulation of a single gene
15-8
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Structural Features of Regulatory
Transcription Factors

Transcription factor proteins contain regions, called
domains, that have specific functions


One domain could be for DNA-binding
Another could provide a binding site for effector molecules

A motif is a domain, or a portion of a domain, that
has a very similar structure in many different proteins

Figure 15.3 depicts several different domain
structures found in transcription factor proteins
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15-9
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Loop
Recognition
helix
Turn
Recognition
helix
(a) Helix–turn–helix motif
(b) Helix–loop–helix motif
The recognition helix recognizes and makes contact
with a base sequence along the major groove of DNA
Hydrogen bonding between an a-helix and nucleotide
bases is one way a transcription factor can bind to DNA
Figure 15.3
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15-10
Composed of one a-helix and
two b-sheets held together by
a zinc (Zn++) metal ion
Two a-helices intertwined
due to leucine motifs
Alternating leucine residues in
both proteins interact (“zip up”),
resulting in protein dimerization
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Zn2+
1
Zinc
finger
Recognition
helix
Leu
Leu
Leu Leucine
side chains
Leu
Leu
(zipper)
Zn2+
2
β sheet
3
Zn2+
Zn2+
Coiled
coil
Zinc
ion
Recognition
helix
4
(c) Zinc finger motif
(d) Leucine zipper motif
Note: Helix-loop-helix motifs can
also mediate protein dimerization
Figure 15.3
Homodimers are formed by two
identical transcription factors;
Heterodimers are formed by two
different transcription factors
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15-11
Enhancers and Silencers

The binding of a transcription factor to an enhancer
increases the rate of transcription


This up-regulation can be 10- to 1,000-fold
The binding of a transcription factor to a silencer
decreases the rate of transcription

This is called down-regulation
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15-12
Enhancers and Silencers

Many response elements are orientation
independent or bidirectional


They can function in the forward or reverse orientation
Most response elements are located within a few
hundred nucleotides upstream of the promoter

However, some are found at various other sites



Several thousand nucleotides away
Downstream from the promoter
Even within introns!
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15-13
TFIID and Mediator

Most regulatory transcription factors do not bind
directly to RNA polymerase

Three common interactions that communicate the
effects of regulatory transcription factors are




1. TFIID-direct or through coactivators
2. Mediator
3. recruiting proteins that affect nucleosome composition
Refer to Figure 15.4 and 15.5
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15-14
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Activator
protein
ON
Coactivator
TFIID
Enhancer
TFIID
Coding sequence
Core
promoter
The activator/coactivator complex recruits TFIID to the core promoter
and/or activates its function. Transcription will be activated.
(a) Transcriptional activation via TFIID
OFF
Repressor
protein
Coding sequence
Silencer
Core
promoter
The repressor protein inhibits the binding of TFIID to the core promoter
or inhibits its function. Transcription is repressed.
(b) Transcriptional repression via TFIID
Figure 15.4
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15-15
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Core
promoter
Core
promoter
ON
Mediator
TFIID
OFF
Mediator
TFIID
RNA polymerase
and general
transcription
factors
RNA polymerase
and general transcription
factors
Coding sequence
Coding sequence
Activator protein
Repressor protein
Enhancer
Silencer
The activator protein interacts with mediator. This enables
RNA polymerase to form a preinitiation complex that can
proceed to the elongation phase of transcription.
(a) Transcriptional activation via mediator



Transcriptional activator stimulates the
function of mediator
This enables RNA pol to form a preinitiation
complex
It then proceeds to the elongation phase of
transcription
The repressor protein interacts with mediator so
that transcription is repressed.
(b) Transcriptional repression via mediator


Transcriptional repressor inhibits the
function of mediator
Transcription is repressed
Figure 15.5
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15-16
Modulation of Regulatory
Transcription Factor Functions


There are three common ways that the function of
regulatory transcription factors can be modulated

1. Binding of a small effector molecule

2. Protein-protein interactions

3. Covalent modification
Refer to Figure 15.6
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15-17
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Hormone
Transcription
factor
The transcription factor
can now bind to DNA
Response
element
Formation of
homodimers and
heterodimers
(a) Binding of a small effector molecule such as a hormone
Transcription
factor
Transcription
factor
Homodimer
(b) Protein–protein interaction
Transcription
factor
PO42–
Inactive
PO42–
Active
(c) Covalent modification such as phosphorylation
Figure 15.6
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15-18
Steroid Hormones and
Regulatory Transcription Factors

Regulatory transcription factors that respond to
steroid hormones are termed steroid receptors


The hormone actually binds to the transcription factor
The ultimate action of a steroid hormone is to affect
gene transcription

Steroid hormones are produced by endocrine glands
 Secreted into the bloodstream
 Then taken up by cells that respond to the hormone
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15-19
Steroid Hormones and
Regulatory Transcription Factors

Cells respond to steroid hormones in different ways

Glucocorticoids


Gonadocorticoids



These influence nutrient metabolism in most cells
 They promote glucose utilization, fat mobilization and protein
breakdown
These include estrogen and testosterone
They influence the growth and function of the gonads
Figure 15.7 shows the stepwise action of
glucocorticoid hormones
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15-20
Heat shock protein
Figure 15.7
Heat shock proteins
released
when
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or display.
hormone binds
Glucocorticoid
Cytoplasm
HSP
90
HSP
90
HSP
90
HSP
90
+
Glucocorticoid
receptor
NLS
Nuclear localization
signal is exposed
Formation of a
homodimer
Nucleus
Core
promoter
Nuclear
pore
5′
Glucocorticoid
Response Elements
These function as
enhancers
GREs are located near
dozens of different
genes, so the hormone
can activate many
genes
3′
A GRA C A
G
T CY T T
TG TY C T
A C ARGA
3′
Target
gene
5′
GRE
Transcription of target
gene is activated
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15-21
The CREB Protein

The CREB protein is another regulatory
transcriptional factor


CREB is an acronym for cAMP response element-binding
CREB protein becomes activated in response to cellsignaling molecules that cause an increase in the
cytoplasmic concentration of cAMP


Cyclic adenosine monophosphate
The CREB protein recognizes a response element with the
consensus sequence 5’–TGACGTCA–3’

This has been termed a cAMP response element (CRE)
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15-22
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Could be a hormone,
neurotransmitter,
growth factor, etc.
Extracellular
signaling
Plasma
molecule
membrane
receptor
G protein
Activates
Activates
Adenylyl
cyclase
Protein
kinase A
cAMP
ATP
Acts as a
second
messenger
ATP
Phosphorylates
2–
Phosphorylated CREB
binds to DNA and
stimulates transcription
Unphosphorylated CREB
can bind to DNA, but
cannot activate RNA pol
Figure 15.8
CREB
protein
dimer
PO4
2–
PO4
Core
promoter
Activates
protein
kinase A
Target gene
CRE
The activity of the CREB protein
15-23
15.2 CHROMATIN REMODELING

ATP-dependent chromatin remodeling refers
to dynamic changes in chromatin structure

These changes range from a few
nucleosomes to large scale changes

Carried out by diverse multiprotein machines
that reposition and restructure nucleosomes
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15-24
Chromatin Structure

The three-dimensional packing of chromatin is an
important parameter affecting gene expression

Chromatin is a very dynamic structure that can
alternate between two conformations

Closed conformation



Chromatin is very tightly packed
Transcription may be difficult or impossible
Open conformation


Chromatin is accessible to transcription factors
Transcription can take place
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15-25



Nucleosomes have been shown to change position
in cells that normally express a particular gene
compared with cells in which the gene is inactive
For b-globin, nucleosome positioning changes in the
promoter region as part of gene activation
A key role of some transcriptional activators is to
orchestrate changes in chromatin structure


Closed conformation
Open conformation
One way is through ATP-dependent chromatin
remodeling.


Energy of ATP hydrolysis is used to drive change in
location and/or composition of nucleosomes
Makes the DNA more or less amenable to transcription
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15-26

ATP-dependent chromatin remodeling

The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
Eukaryotes have multiple families of
chromatin remodelers;
SWI/SNF
ISWI
INO80
Mi-2
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ATP-dependent
chromatin-remodeling
complex
or
Change in the relative positions
of a few nucleosomes
Figure 15.9a
Change in the spacing
of nucleosomes over a
long distance
These effects may significantly alter
gene expression
(a) Change in nucleosome position
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15-27

ATP-dependent chromatin remodeling
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ATP-dependent
chromatin-remodeling
complex
Histone octamers are removed.
(b) Histone eviction
ATP-dependent
chromatin-remodeling
Variant histones complex
(c) Replacement with variant histones
Figure 15.9b and c
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15-28

The five histone genes are moderately repetitive


Human genome contains over 70 histone genes



H1, H2A, H2B, H3 and H4
Most encode standard histones
A few of these genes have accumulated mutations that
alters the amino acid sequence
 These are termed variants
Some histone variants are incorporated into a
subset of nucleosomes to create specialized
chromatin

See Table 15.1
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15-29
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15-30
Histone Code

Over 50 enzymes have been identified in mammals
that selectively modify the amino terminal tails of
histones


acetylation, methylation and phosphorylation are common
(see Figure 15.10)
These modifications affect the level of transcription


May influence interactions between nucleosomes
Occur in patterns that are recognized by proteins



Called the histone code
The pattern of modifications specify alterations to be made to
chromatin structure
These proteins bind based on the code and affect transcription
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15-31
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ac
5
Lys
Amino-terminal tail
p
p
ac
Lys
Ser
ac
Lys
10
5
15
Ser
m
5
H2B
H2A
p
Lys
ac Lys
Lys
10
m ac
Arg Lys
m
m
ac
m
ac p
Lys
15
LysSer
10
ac
20 Lys
20
Globular domain
Arg
Lys
Ser 15
10
ac
Lys
ac
5
ac
ac
15 Lys
ac
m
Lys
20
Ser
Lys
H4
20
H3
(a) Examples of histone modifications
Core histone
protein
COCH3
Histone
acetyltransferase
Acetyl
group
COCH3
Histone
deacetylase
COCH3
DNA is less tightly bound
to the histone proteins
(b) Effect of acetylation
Figure 15.10
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15-32
Chromatin Immunoprecipitation
Sequencing


Also known as ChIP-Seq
Allows determination of:




Where nucleosomes are located
Where histone variants are found
Where covalent modifications of histones occur
See Figure 15.11
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15-33
Chromatin Immunoprecipitation Sequencing
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Core histone
proteins
Nonhistone
proteins
Cell nucleus
While the DNA is still in the cell nucleus, expose
cells to a reagent, such as formaldehyde, that
crosslinks proteins to DNA.
Covalent
crosslink
Break open cells and cell nuclei, and expose to
micrococcal nuclease that cuts the linker regions
between nucleosomes. DNA that is crosslinked
to proteins generally remains intact.
Bead
Antibody
Add antibodies that recognize a specific protein
or proteins. In this example, the antibodies
recognize the core histone proteins. The
antibodies are attached to large beads.
DNA and protein not
attached to antibody
Core histone protein
attached to antibody
Figure 15.11
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15-34
Chromatin Immunoprecipitation Sequencing
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Centrifuge at a speed that causes the
beads and anything attached to the beads
to form a pellet at the bottom of the tube.
DNA and proteins not attached to the
beads remain in the supernatant.
Supernatant: DNA and proteins not attached
to the beads
Pellet: DNA and proteins attached to the beads
Discard supernatant. Expose pellet to a reagent
that breaks the crosslinks between the proteins
and DNA.
DNA that had been wrapped
around core histones
Core histones
Purify DNA fragments with a size of approximately
150 bp by gel electrophoresis. Add linkers to the
ends of the DNA.
~150 bp
Linker
Amplify by PCR, using primers that are
complementary to the linkers, and subject to
DNA sequencing. These methods are
described in Chapter 18.
Figure 15.11
The end result is a large collection of DNA sequences. These sequences
can be matched up with sequences in the genome to determine which
segments in a genome are found within nucleosomes.
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15-35
Chromatin Immunoprecipitation
Sequencing has revealed a common
pattern of nucleosome organization
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–2
–1 NFR +1
+2
+3
NFR
Nucleosome
positions:
DNA
Transcriptional start site
Transcriptional termination site
A nucleosome-free region (NFR) is found at the beginning and end of many
genes. Nucleosomes tend to be precisely positioned near the beginning and
end of a gene, but are less regularly distributed elsewhere.
Figure 15.12
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15-36
Transcriptional Activation
Silent: Many genes are flanked by nucleosome-free regions (NFR)
and well-positioned nucleosomes.
-2
-1
NFR
+1
+2
NFR
Enhancer
Transcriptional
start site
Transcriptional
termination site
Binding of activators:
Activator proteins bind to enhancer
sequences. The enhancers may
be close to the transcriptional start
site (as shown here) or they may
be far away.
Activator
-2
-1
Enhancer
+1
+2
Chromatin remodeling and
histone modification:
Activator proteins recruit chromatin
remodeling complexes, such as
SWI/SNF, and histone modifying
enzymes such as histone
acetyltransferase. Nucleosomes
may be moved, and histones may
be evicted. Some histones are
subjected to covalent modification,
such as acetylation.
Figure 15.13
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15-37
Transcriptional Activation
Histone acetyltransferase
-2
+2
AC
SWI/
SNF
AC
AC
AC
AC
Formation of the pre-initiation
complex:
General transcription factors and
RNA polymerase II are able to bind
to the core promoter and form a
pre-initiation complex.
-2
+2
AC
AC
AC
AC
AC
Elongation:
During elongation, histones ahead
of the open complex are covalently
modified by acetylation and evicted or
partially displaced. Behind the
open complex, histones are
deacetylated and become tightly
bound to the DNA.
Pre-initiation complex
Deacetylated histones
-2
-1
+1
+2
Pre-mRNA
AC
AC
AC
AC
Open complex
Evicted histone
proteins
Figure 15.13
Chaperone
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15-38
15.3 DNA Methylation



DNA methylation is a change in chromatin structure
that silences gene expression
Carried out by the enzyme DNA methyltransferase
It is common in some eukaryotic species, but not all


Yeast and Drosophila have little DNA methylation
Vertebrates and plants have abundant DNA methylation


In mammals, ~ 2 to 7% of the DNA is methylated
Refer to Figure 15.14
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15-39
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5′
3′
C
G
NH2
N3
2
O
H
4
5
3′
Cytosine
5′
(b) Unmethylated
H
H
5′
DNA methyltransferase
3′
CH3
C
G
Only one strand is
methylated
G
C
3′
NH2
4
C
6
1
N
N3
G
5′
(c) Hemimethylated
CH3
5
5-methylcytosine
2
O
1
6
N
H
5′
CH3
H
(a) The methylation of cytosine
3′
C
G
3′
G
C
Both strands are
methylated
CH3
5′
(d) Fully methylated
Figure 15.14
15-40

DNA methylation usually inhibits the transcription of
eukaryotic genes


Especially when it occurs in the vicinity of the promoter
In vertebrates and plants, many genes contain
CpG islands near their promoters

These CpG islands are 1,000 to 2,000 nucleotides long


In housekeeping genes



Contain high number of CpG sites
The CpG islands are unmethylated
Genes tend to be expressed in most cell types
In tissue-specific genes



The expression of these genes may be silenced by the
methylation of CpG islands
Methylation may influence the binding of transcription factors
Methyl-CpG-binding proteins may recruit factors that lead to
compaction of the chromatin
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15-41
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Enhancer
CpG island
Core promoter
Coding sequence
Activator
protein
Methylation
Transcriptional
activator binds to
unmethylated DNA
CH3
CH3
CH3
CH3
CH3
CH3
Methyl groups block the
binding of an activator protein
to an enhancer element.
This would inhibit the
initiation of transcription
(a) Methylation inhibits the binding of an activator protein.
Figure 15.15a
Transcriptional silencing via methylation
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15-42
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CH3
CpG island
CH3
CH3
CH3
CH3
Core promoter
CH3
CH3
CH3
CH3
Chromatin
in an open
conformation
A methyl-CpG-binding protein
binds to the methylated
CpG island.
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Methyl-CpG
Binding protein
Chromatin
in a closed
conformation
CH3
CH3 CH3
The methyl-CpG-binding protein
recruits other proteins, such as
histone deacetylase, that convert
the chromatin to a closed
conformation.
CH3
CH3
CH3
CH3
CH3
CH3
Histone
deacetylase
(b) Methyl-CpG-binding protein recruits other proteins that change the
chromatin to a closed conformation.
Figure 15.15b
Transcriptional silencing via methylation
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15-43
DNA Methylation is Heritable


Methylated DNA sequences are inherited during
cell division
May explain genomic imprinting (Chapter 5)



Specific genes are methylated in gametes from mother or
father
Pattern of one copy of the gene being methylated and the
other not is maintained in the resulting offspring
Figure 15.16 illustrates a model explaining how
methylation is passed from mother to daughter cell
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15-44
Figure 15.16
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5
3′
C
G
G
C
3′
5′
An infrequent and
highly regulated event
de novo methylation
5′
3′
CH3
C
G
G
C
CH3
3′
5′
DNA replication
Hemimethylated
DNA
5′
3′ 5′
CH3
C
G
3′
C
G
G
C
3′
CH3
5′ 3′
5′
Maintenance
methylation
5′
Fully methylated
DNA
3′ 5′
CH3
3′
G
C
C
G
G
C
3′
CH3
CH3
5′ 3′
C
G
G
C
DNA methyltransferase
converts hemimethylated to fullymethylated DNA
An efficient and routine
event occurring in
vertebrate and plant cells
CH3
5′
15-45
15.4 INSULATORS


Since eukaryotic gene regulation can occur over
long distances, it is important to limit regulation to
one particular gene, but not to neighboring genes
Insulators are segments of DNA that insulates a
gene from the regulatory effects of other genes



Some act as barriers to chromatin remodeling
Others block the effects of enhancers
See Figure 15.17
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15-46
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Proteins that bind
to insulators
ac
ac
ac
ac
Nonacetylated
DNA
Insulator
ac
ac
ac
ac
Gene within an
acetylated region
of DNA
Nonacetylated
DNA
Insulator
(a) Insulators as a barrier to changes in chromatin structure
Protein bound
to an insulator
Gene A
The insulator prevents
the enhancer for gene A
from activating the
expression of gene B.
Enhancer
Gene B
Insulator
(b) Insulator that blocks the effects of a neighboring enhancer
Figure 15.17
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15-47
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
OFF
Igf2 gene is not stimulated by the enhancer.
H19
Enhancer
Enhancer
H19
ICR
(Insulator)
Igf2
DMR
No methylation
ICR
(Insulator)
Igf2
CTC-binding
proteins
DMR
ICR
(Insulator)
Methylation
Enhancer
H19
ON
Igf2
CH3
CH3 CH3
DMR
CH3
CH3 CH3
Methylation prevents the binding of CTC-binding proteins.
Igf2 gene can be stimulated by the enhancer.
Insulators may function by creating a loop in the DNA. This loop formation
can be blocked by methylation, which may play a role in imprinting.
Figure 15.18
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15-48
15.5 REGULATION OF RNA
PROCESSING AND TRANSLATION

So far, we have discussed various mechanisms
that regulate the level of gene transcription

In eukaryotic species, it is also common for gene
expression to be regulated at the RNA level

Refer to Table 15.2
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15-49
15-50
Alternative Splicing


One very important biological advantage of introns in
eukaryotes is the phenomenon of alternative splicing
Alternative splicing refers to the phenomenon that
pre-mRNA can be spliced in more than one way



Alternatively splicing produces two or more polypeptides
with different amino acid sequences
In most cases, large sections of the coding regions are the
same, resulting in alternative versions of a protein that
have similar functions
Nevertheless, there will be enough differences in amino
acid sequences to provide each polypeptide with its own
unique characteristics
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15-51
Alternative Splicing

The degree of splicing and alternative splicing
varies greatly among different species

Baker’s yeast contains about 6,300 genes

~ 300 (i.e., 5%) encode mRNAs that are spliced


Only a few of these 300 have been shown to be alternatively spliced
Humans contain ~ 25,000 genes

Most of these encode mRNAs that are spliced


It is estimated that about 70% are alternatively spliced
Note: Certain mRNAs can be alternatively spliced to produce dozens
of different mRNAs
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15-52
Alternative Splicing

Figure 15.19 considers an example of alternative
splicing for a gene that encodes a-tropomyosin


This protein functions in the regulation of cell contraction
It is found in




Smooth muscle cells (uterus and small intestine)
Striated muscle cells (cardiac and skeletal muscle)
Also in many types of nonmuscle cells at low levels
The different cells of a multicellular organism regulate
contractibility in subtly different ways

One way to accomplish this is to produce different forms of
a-tropomyosin by alternative splicing
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15-53
Found in the mature mRNA
from all cell types
Not found in all
mature mRNAs
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Intron
5′
1
2
α-tropomyosin pre-mRNA
Exon
3
4
5
6
7
8
9
10
11
12
13 14
3′
Constitutive exons
Alternative
splicing
5′
1
2
4
5
6
Alternative exons
3′
8
9
10 14
8
9
10 11 12
Smooth muscle cells
or
5′
1
3
4
5
6
3′
Striated muscle cells
These alternatively spliced versions of a-tropomyosin vary in
function to meet the needs of the cell type in which they are found
Figure 15.19
Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
15-54
Alternative Splicing

Alternative splicing is not a random event


It involves proteins known as splicing factors


The specific pattern of splicing is regulated in a given cell
These play a key role in the choice of splice sites
One example of splicing factors are the SR proteins

At their C-terminal end, they have a domain that is rich in
serine (S) and arginine (R)


It is involved in protein-protein recognition
At their N-terminal end, they have an RNA-binding domain
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15-55

The spliceosome recognizes the 5’ and 3’ splice sites
and removes the intervening intron

Refer to Chapter 12

Splicing factors modulate the ability of spliceosomes
to recognize or choose the splice sites

This can occur in two ways

1. Some splicing factors inhibit the ability of a spliceosome
to recognize a splice site


Refer to Figure 15.20a
2. Some splicing factors enhance the ability of a
spliceosome to recognize a splice site

Refer to Figure 15.20b
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15-56
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Alternative
splicing
5′ 3′
1
5′
5′ 3′
Splice
junctions
5′ 3′
2
3
4
The spliceosome
recognizes all the
splice junctions.
5′
1
2
3
All 4 exons are contained
within the mRNA.
4
3′
5′ 3′
5′
1
5′ 3′
2
5′
3
4
3′
A splicing repressor prevents the
recognition of a 3′-splice junction.
The next 3′-splice junction that
precedes exon 3 will be chosen.
Splicing
repressor
3′
S plic e
ju n ctio n s
5′ 3′
1
3
4
3′
Exon 2 is skipped and not
included in the mRNA.
(a) Splicing repressors
Known as exon
skipping
Figure 15.20 The role of splicing factors during alternative splicing
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15-57
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Alternative
splicing
These 2 splice junctions
are not readily recognized
by the spliceosome.
5′
5′
3′
1
5′
2
3′
5′
Splice
junctions
3′
3
4
The spliceosome only
recognizes 4 of the
6 splice junctions.
5′
1
2
4
3′
Exon 3 is not included in the mRNA.
3′
5′
5′
3′
5′
1
3′
2
1
Splice
junctions
3′
3
Splicing
enhancer
5′
5′
4
3′
The binding of splicing enhancers
promotes the recognition of
poorly recognized junctions. All 6
junctions are recognized.
2
3
4
3′
Exon 3 is included in the mRNA.
(b) Splicing enhancers
Figure 15.20 The role of splicing factors during alternative splicing
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15-58
Stability of mRNA

The stability of eukaryotic mRNA varies considerably


Several minutes to several days or even months
The stability of mRNA can be regulated so that its
half-life is shortened or lengthened

This will greatly influence the mRNA concentration


And consequently gene expression
Factors that can affect mRNA stability include


1. Length of the polyA tail
2. Destabilizing elements
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15-59

1. Length of the polyA tail


Most newly made mRNA have a polyA tail that is about
200 nucleotides long
It is recognized by polyA-binding protein



Which binds to the polyA tail and enhances stability
As an mRNA ages, its polyA tail is shortened by the
action of cellular exonucleases
The polyA-binding protein can no longer bind if the polyA
tail is less than 10 to 30 adenosines long

The mRNA will then be rapidly degraded by exo- and
endonucleases
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15-60

2. Destabilizing elements


Found in mRNAs that have short half-lives
These elements can be found anywhere in the mRNA

However, they are most commonly at the 3’ end between the stop
codon and the polyA tail
AU-rich element (ARE)
Recognized and bound by cellular proteins
These proteins influence mRNA degradation
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PolyA tail
ARE
mRNA Start codon
5′
Stop codon
AUG
5′-UTR
AUUUA
AAAAAAAA 3′
3′-UTR
5’-untranslated region
Figure 15.21
Protein binding
to ARE
3’-untranslated region
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15-61
Double-stranded RNA and Gene
Silencing

Double-stranded RNA can silence the expression of
certain genes


This discovery was made from research in plants and the
nematode Caenorhabditis elegans
Using cloning techniques, it is possible to introduce
cloned genes into the genomes of plants

When cloned genes were introduced in multiple copies, the
expression of the gene was often silenced

This may be due to the formation of double-stranded RNA. See
Figure 15.22
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15-62
Promoter of endogenous
plant gene uses the top
strand as the template
strand and transcribes
to the left.
Endogenous
plant gene
PE
5′
3′
Plant
chromosomal
DNA
A cloned gene randomly
inserts into the plant
chromosome, next to an
endogenous promoter.
Cloned gene
5′
3′
3′
5′
PE
PC
Promoter for cloned gene
uses bottom strand as the
template strand and
transcribes to the right.
This event will silence the
expression of the cloned gene
3′
5′
Transcription occurs from
both promoters, producing
complementary RNAs that
form a double-stranded
structure.
5′
3′
Strand transcribed
from cloned gene
promoter
3′
5′
Strand transcribed
from endogenous
plant promoter
Figure 15.22 Gene insertion leading to the production of double-stranded RNA
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15-63
Experiment 15A: Double-stranded
RNA and Gene Silencing

Evidence for mRNA degradation via double-stranded
RNA came from studies in C. elegans

Injection of antisense RNA (i.e., RNA complementary to a
specific mRNA) into oocytes silences gene expression



Surprisingly, injection of double-stranded RNA was 10 times more
potent at inhibiting the expression of the corresponding mRNA
Also, the effects of antisense RNA persisted for a very long time
This led Andrew Fire and Craig Mello to investigate how
injection of RNA inhibits mRNA
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15-64
The Goal

Understand how the experimental injection of
RNA was responsible for the silencing of
particular mRNAs
Achieving the Goal

Refer to Figure 15.23
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15-65
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Conceptual level
Experimental level
1. Make sense and antisense mex-3 RNA
in vitro using cloned genes for mex-3
with promoters on either side of the
Gene. RNA polymerase and nucleotides
are added to synthesize the RNAs.
Sense RNA
Promoter
Add RNA
polymerase and
nucleotides to
cloned genes.
Sense
RNA
mex-3
gene
RNA
polymerase
Antisense RNA
2. Inject either mex-3 antisense RNA or a
mixture of mex-3 sense and antisense
RNA into the gonads of C. elegans. This
RNA is taken up by the eggs and early
embryos. As a control, do not inject any
RNA.
Antisense RNA or a
mixture of sense and
antisense RNA
Antisense RNA
Promoter]
Single row of eggs
Add labeled
probe.
3. Incubate and then subject early embryos
to in situ hybridization. In this method,
a labeled probe is added that is
complementary to mex-3 mRNA. If cells
express mex-3, the mRNA in the cells
will bind to the probe and become
labeled. After incubation with a labeled
probe, the cells are washed to remove
unbound probe.
Labeled probe
Embryo
mex-3 mRNA
4. Observe embryos under the
microscope.
Figure 15.23
15-66
The Data
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Control
Injected with mex-3
antisense RNA
Photos reprinted by permission from
Macmillan Publishers Ltd. A. Fire, S. Xu, M.K.
Montgomery, et al. (1998) Potent and specific
genetic interference by double-stranded RNA
in Caenorhabditis elegans. Nature. 391:6669,
806–811.
Injected with both mex-3 sense
and antisense RNA
Figure 15.23
15-67
Interpreting the Data
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Control
Injected with mex-3
antisense RNA
Photos reprinted by permission from
Macmillan Publishers Ltd. A. Fire, S. Xu, M.K.
Montgomery, et al. (1998) Potent and specific
genetic interference by double-stranded RNA
in Caenorhabditis elegans. Nature. 391:6669,
806–811.
mex-3 is expressed at high
levels in the control, lower levels
in cells injected with antisense
RNA and completely degraded
in cells injected with doublestranded RNA. This shows that
double-stranded RNA is more
potent at silencing gene
expression than antisense RNA.
They termed this phenomenon
RNA interference or RNAi
Injected with both mex-3 sense
and antisense RNA
Figure 15.23
15-68
RNA Is Interference Mediated by
Micro RNAs

microRNAs (miRNAs) or short-interfering RNAs
(siRNAs) cause RNA interference

encoded by genes in eukaryotic organisms


genes do not encode a protein
give rise to small RNA molecules, typically 21 to 23 nucleotides

Silence expression of specific mRNAs

In humans, approximately 200 genes encoding miRNAs
have been identified

A proposed mechanism for RNAi is shown in Figure 15.24
15-69
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Pre-miRNA or pre-siRNA
transcribed from a gene
Hairpin structure
Mechanism of RNA
interference
5′
Dicer
The double-stranded RNA is
cut by Dicer to yield a double-stranded
RNA about 21 to 23
bp long.
3′
The double-stranded RNA is
recognized by a protein that
associates with other proteins
to form the RNA-induced
silencing complex (RISC).
One of the RNA strands is
degraded.
RISC
The RISC recognizes specific
cellular mRNAs, due to
complementarity.
Complementary region
between cellular mRNA
and miRNA or siRNA
RISC
siRNA
miRNA
OR
Figure 15.24
The cellular mRNA
is degraded.
(High complementarity)
The mRNA is unable
to be translated.
(Low complementarity)
15-70
Benefits of RNA interference


Presents a newly identified form of gene regulation
May offer a defense mechanism against certain
viruses



RNA viruses that have a double-stranded RNA genome
RNA viruses that produce a double-strand RNAs during
their life cycle
May play a role in silencing certain transposable
elements

Random insertion may place an element near a cellular promoter
which will produce a silencing RNA
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15-71
Initiation Factors and the Rate of
Translation

Modulation of translation initiation factors is widely
used to control fundamental cellular processes

Under certain conditions, it is advantageous for a
cell to stop synthesizing proteins

Viral infection


So that the virus cannot manufacture viral proteins
Starvation

So that the cell conserves resources
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15-72

The phosphorylation of initiation factors has been
found to affect translation in eukaryotic cells


The functions of these two factors are modulated by
phosphorylation in opposite ways



Two initiation factors appear to play a central role in
controlling the initiation of translation
 eIF2 and eIF4F
Phosphorylation of eIF2a inhibits translation
Phosphorylation of eIF4F increases the rate of translation
Figure 15.25 shows the events leading to
translational inhibition by eIF2a
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15-73
Conditions: Viral infection
Nutrient deprivation
Heat shock
Toxic heavy metals
eIF2α
proten
kinase
Inactive
Active
2–
PO 4
2–
PO 4
2–
PO 4
Required if eIF2 is to
promote binding of the
initiator tRNAmet to the 40S
subunit
Active
Inactive
Met
40S ribosomal
subunit
Figure 15.25
Translation is inhibited because the initiator
tRNAMet does not bind to the 40S subunit.
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15-74

eIF4F provides another way to control translation


It regulates the binding of mRNA to the ribosomal initiation
complex
eIF4F is stimulated by phosphorylation

Conditions that increase its phosphorylation include
signaling molecules that promote cell proliferation


Growth factors and insulin, for example
Conditions that decrease its phosphorylation include heat
shock and viral infection
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15-75
Iron Assimilation and Translation

Regulation of iron assimilation provides an example
how the translation of specific mRNAs is modulated

Iron is an essential element for the survival of living
organisms


It is required for the function of many different enzymes
The assimilation of iron is depicted in Figure 15.26
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15-76
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Fe3+
Protein that carries iron
through the bloodstream
Transferrin
Transferrin
receptor
Endocytic vesicle
Endocytosis
Fe3+
binds to
cellular
enzymes
A hollow spherical protein
Prevents toxic buildup of
too much iron in the cell
Fe3+
Fe3+
Fe3+
Ferritin
Iron (Fe3+)
is released
into cytosol
Fe3+ stored
within ferritin
Figure 15.26
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15-77

Iron is a vital yet potentially toxic substance


So mammalian cells have evolved an interesting way to
regulate iron assimilation
An RNA-binding protein known as the iron regulatory
protein (IRP) plays a key role

It influences both the ferritin mRNA and the transferrin
receptor mRNA

This protein binds to a regulatory element within the mRNA
known as the iron response element (IRE)



An IRE is found in the 5’-UTR of ferritin mRNA
An IRE is also found in the 3’-UTR of transferrin receptor mRNA
Regulation of iron assimilation is shown in Figure 15.27
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15-78
Figure 15.27 (a) Regulation of ferritin mRNA
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High iron
Low iron
Ferritin
mRNA
Iron response
element (IRE)
Ferritin
protein
Fe3+
Inactive IRP
Iron regulatory
protein (IRP)
5′
AAAAA3′
5′-UTR
Stop codon
Start codon
IRP binds to the IRE and inhibits translation.
AAAAA3′
5′
IRE
Start codon
Stop codon
IRP binds iron and is released from the IRE; translation proceeds.
(a) Regulation of ferritin mRNA
Ferritin translation is inhibited by low iron, but not by high iron
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15-79
Figure 15.27 (b) Regulation of transferrin receptor mRNA
Increased stability of
mRNA means more
translation
mRNA is degraded and
cannot be translated
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Transferrin
receptor
protein
Low iron
High iron
Fe3+
Inactive IRP
IRE
Iron
regulatory
protein (IRP)
5′
AAAAA3′
Start codon
Stop codon
3′ -UTR
AAAAA3′
5′
Start codon
Stop codon
IRE
Transferrin receptor mRNA
Endonuclease
IRP binds to the IRE and enhances the stability of the mRNA.
IRP binds iron and is released from the IRE; the mRNA is degraded.
(b) Regulation of transferrin receptor mRNA
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15-80