Chapter 15
Lecture Outline
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
INTRODUCTION
n
n
Eukaryotic organisms derive many benefits from
regulating their genes
For example
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-2
INTRODUCTION
n
Gene regulation is necessary to ensure
n
1. Expression of genes in an accurate pattern during the
various developmental stages of the life cycle
n
n
2. Differences among distinct cell types
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-3
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
n
n
Transcription factors are proteins that influence the
ability of RNA polymerase to transcribe a given gene
There are two main types
n
General transcription factors
n
n
n
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
n
n
Serve to regulate the rate of transcription of target genes
They influence the ability of RNA pol to begin transcription of a
particular gene
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-5
n
Regulatory transcription factors recognize cis
regulatory elements located near the core promoter
n
n
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
n
A regulatory protein that increases the rate of
transcription is termed an activator
n
n
A regulatory protein that decreases the rate of
transcription is termed a repressor
n
n
The sequence it binds is called an enhancer
The sequence it binds is called a silencer
Refer to Figure 15.2
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-6
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-7
n
Most Eukaryotic genes are regulated by many factors
n
n
This is known as combinatorial control
Common factors contributing to combinatorial control are:
n
n
n
One or more activator proteins may stimulate transcription
One or more repressor proteins may inhibit transcription
Activators and repressors may be modulated by:
n
n
n
n
n
Regulatory proteins may alter nucleosomes near the promoter
DNA methylation may inhibit transcription
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Structural Features of Regulatory
Transcription Factors
n
Transcription factor proteins contain regions, called
domains, that have specific functions
n
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-9
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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 α-helix and nucleotide
bases is one way a transcription factor can bind to DNA
Figure 15.3
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-10
Composed of one α-helix and
two β-sheets held together by
a zinc (Zn++) metal ion
Two α-helices intertwined
due to leucine motifs
Alternating leucine residues in
both proteins interact ( zip up ),
resulting in protein dimerization
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-11
Enhancers and Silencers
n
The binding of a transcription factor to an enhancer
increases the rate of transcription
n
n
This up-regulation can be 10- to 1,000-fold
The binding of a transcription factor to a silencer
decreases the rate of transcription
n
This is called down-regulation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-12
Enhancers and Silencers
n
Many response elements are orientation
independent or bidirectional
n
n
They can function in the forward or reverse orientation
Most response elements are located within a few
hundred nucleotides upstream of the promoter
n
However, some are found at various other sites
n
n
n
Several thousand nucleotides away
Downstream from the promoter
Even within introns!
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-13
TFIID and Mediator
n
n
Most regulatory transcription factors do not bind
directly to RNA polymerase
Three common interactions that communicate the
effects of regulatory transcription factors are
n
n
n
n
1. TFIID-direct or through coactivators
2. Mediator
3. recruiting proteins that affect nucleosome composition
Refer to Figure 15.4 and 15.5
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-14
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-15
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
n
n
n
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
n
n
Transcriptional repressor inhibits the
function of mediator
Transcription is repressed
Figure 15.5
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-16
Modulation of Regulatory
Transcription Factor Functions
n
n
There are three common ways that the function of
regulatory transcription factors can be modulated
n
1. Binding of a small effector molecule
n
2. Protein-protein interactions
n
3. Covalent modification
Refer to Figure 15.6
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-17
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-18
Steroid Hormones and
Regulatory Transcription Factors
n
Regulatory transcription factors that respond to
steroid hormones are termed steroid receptors
n
n
The hormone actually binds to the transcription factor
The ultimate action of a steroid hormone is to affect
gene transcription
n
Steroid hormones are produced by endocrine glands
n  Secreted into the bloodstream
n  Then taken up by cells that respond to the hormone
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-19
Steroid Hormones and
Regulatory Transcription Factors
n
Cells respond to steroid hormones in different ways
n
Glucocorticoids
n
n
Gonadocorticoids
n
n
n
These influence nutrient metabolism in most cells
n  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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-20
Heat shock protein
Figure 15.7
Heat shock proteins
released
when
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction
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
3′
A GRA C A
T CY T G T
TG TY C T
A C ARGA
3′
These function as
enhancers
GREs are located near
dozens of different
genes, so the hormone
can activate many
genes
Target"
gene
5′
GRE
Transcription of target
gene is activated
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-21
The CREB Protein
n
The CREB protein is another regulatory
transcriptional factor
n
n
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
n
n
Cyclic adenosine monophosphate
The CREB protein recognizes a response element with the
consensus sequence 5 –TGACGTCA–3
n
This has been termed a cAMP response element (CRE)
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Could be a hormone,
neurotransmitter,
growth factor, etc.
Extracellular"
signaling"
Plasma"
molecule
membrane"
receptor
G protein
Activates
Activates
Adenylyl"
cyclase"
Protein "
kinase A
Activa
tes
cAMP
ATP
Acts as a
second
messenger
ATP
Phosphorylates
2–
PO4
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
2–
PO4
Core"
promoter"
Activates
protein
kinase A
Target gene
CRE
The activity of the CREB protein
15-23
15.2 CHROMATIN REMODELING
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-24
Chromatin Structure
n
n
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
n
Closed conformation
n
n
n
Chromatin is very tightly packed
Transcription may be difficult or impossible
Open conformation
n
n
Chromatin is accessible to transcription factors
Transcription can take place
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-25
n
n
n
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 β-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
n
n
Closed conformation
Open conformation
One way is through ATP-dependent chromatin
remodeling.
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-26
n
ATP-dependent chromatin remodeling
n
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-27
n
ATP-dependent chromatin remodeling
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-28
Histone Code
n
Over 50 enzymes have been identified in mammals
that selectively modify the amino terminal tails of
histones
n
n
acetylation, methylation and phosphorylation are common
(see Figure 15.10)
These modifications affect the level of transcription
n
n
May influence interactions between nucleosomes
Occur in patterns that are recognized by proteins
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-31
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
ac
5
Lys
Amino-terminal tail
p
p
ac
Lys
Ser
ac
Lys
Lys
10
15
Ser
m
5
H2B
ac Lys
Lys
10
m ac
Arg Lys
m
m
ac
m
ac p
20
H2A
p
Lys
Lys
15
LysSer
10
ac
Lys
20
Globular domain
Arg
Lys
Ser 15
10
ac
5
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-32
Chromatin Immunoprecipitation
Sequencing has revealed a common
pattern of nucleosome organization
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
–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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-38
15.3 DNA Methylation
n
n
n
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
n
n
Yeast and Drosophila have little DNA methylation
Vertebrates and plants have abundant DNA methylation
n
n
In mammals, ~ 2 to 7% of the DNA is methylated
Refer to Figure 15.14
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-39
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′
3′
C
G
NH2
N3
2
O
H
4
5
5′
(b) Unmethylated
H
N
C
3′
Cytosine
6
1
G
H
5′
DNA methyltransferase
3′
CH3
C
G
2
O
4
C
3′
NH2
N3
Only one strand is
methylated
G
5
5′
(c) Hemimethylated
CH3
5-methylcytosine
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
n
DNA methylation usually inhibits the transcription of
eukaryotic genes
n
n
Especially when it occurs in the vicinity of the promoter
In vertebrates and plants, many genes contain
CpG islands near their promoters
n
These CpG islands are 1,000 to 2,000 nucleotides long
n
n
In housekeeping genes
n
n
n
Contain high number of CpG sites
The CpG islands are unmethylated
Genes tend to be expressed in most cell types
In tissue-specific genes
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-41
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-42
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
CH3"
CH3"
CH3"
CH3"
CH3"
CH3"
CH3"
CH3"
CpG island
Core promoter
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-43
DNA Methylation is Heritable
n
n
Methylated DNA sequences are inherited during
cell division
May explain genomic imprinting (Chapter 5)
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-44
Figure 15.16
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5ʹ′
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
15.5 REGULATION OF RNA
PROCESSING AND TRANSLATION
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-49
15-50
Alternative Splicing
n
n
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
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-51
Alternative Splicing
n
The degree of splicing and alternative splicing
varies greatly among different species
n
Baker s yeast contains about 6,300 genes
n
~ 300 (i.e., 5%) encode mRNAs that are spliced
n
n
Only a few of these 300 have been shown to be alternatively spliced
Humans contain ~ 25,000 genes
n
Most of these encode mRNAs that are spliced
n
n
It is estimated that about 70% are alternatively spliced
Note: Certain mRNAs can be alternatively spliced to produce dozens
of different mRNAs
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-52
Alternative Splicing
n
Figure 15.19 considers an example of alternative
splicing for a gene that encodes α-tropomyosin
n
n
This protein functions in the regulation of cell contraction
It is found in
n
n
n
n
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
n
One way to accomplish this is to produce different forms of
α-tropomyosin by alternative splicing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-53
Found in the mature mRNA
from all cell types
Not found in all
mature mRNAs
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Intron
5′
1
2
α-tropomyosin pre-mRNA
Exon
3
4
5
6
7
8
9
10
11
12
1
2
4
5
6
3′
Constitutive exons
Alternative"
splicing
5′
13 14
Alternative exons
8
9
1 0 14
8
9
1 0 1 1 12
Smooth muscle cells
3′
or
5′
1
3
4
5
6
3′
Striated muscle cells
These alternatively spliced versions of α-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 α-tropomyosin pre-mRNA can be spliced
15-54
Alternative Splicing
n
Alternative splicing is not a random event
n
n
It involves proteins known as splicing factors
n
n
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
n
At their C-terminal end, they have a domain that is rich in
serine (S) and arginine (R)
n
n
It is involved in protein-protein recognition
At their N-terminal end, they have an RNA-binding domain
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-55
n
The spliceosome recognizes the 5 and 3 splice
sites and removes the intervening intron
n
n
n
Refer to Chapter 12
Splicing factors modulate the ability of spliceosomes
to recognize or choose the splice sites
This can occur in two ways
n
1. Some splicing factors inhibit the ability of a spliceosome
to recognize a splice site
n
n
Refer to Figure 15.20a
2. Some splicing factors enhance the ability of a
spliceosome to recognize a splice site
n
Refer to Figure 15.20b
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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′
3′
5′ 3′
5′
1
5′ 3′
2
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
5′
S p lic e
ju n c t i o 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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-57
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-58
Stability of mRNA
n
The stability of eukaryotic mRNA varies considerably
n
n
Several minutes to several days or even months
The stability of mRNA can be regulated so that its
half-life is shortened or lengthened
n
This will greatly influence the mRNA concentration
n
n
And consequently gene expression
Factors that can affect mRNA stability include
n
n
1. Length of the polyA tail
2. Destabilizing elements
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-59
n
1. Length of the polyA tail
n
n
Most newly made mRNA have a polyA tail that is about
200 nucleotides long
It is recognized by polyA-binding protein
n
n
n
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
n
The mRNA will then be rapidly degraded by exo- and
endonucleases
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-60
n
2. Destabilizing elements
n
n
Found in mRNAs that have short half-lives
These elements can be found anywhere in the mRNA
n
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
PolyA tail"
ARE"
mRNA Start codon"
5′
AUG"
Stop codon"
UAA"
5′-UTR"
AAAAAAAA 3′
3′-UTR"
5 -untranslated region
Figure 15.21
AUUUA"
Protein binding"
to ARE"
3 -untranslated region
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-61
Double-stranded RNA and Gene
Silencing
n
Double-stranded RNA can silence the expression of
certain genes
n
n
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
n
When cloned genes were introduced in multiple copies, the
expression of the gene was often silenced
n
This may be due to the formation of double-stranded RNA. See
Figure 15.22
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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′
3′
5′
Plant"
chromosomal"
DNA
A cloned gene randomly"
inserts into the plant"
chromosome, next to an"
endogenous promoter.
Cloned gene
5′
3′
PE
PC
3′
5′
Promoter for cloned gene"
uses bottom strand as the"
template strand and"
transcribes to the right.
Transcription occurs from"
both promoters, producing"
This event will silence the
complementary RNAs that"
expression of the cloned gene
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-63
RNA Is Interference Mediated by
Micro RNAs
n
microRNAs (miRNAs) or short-interfering RNAs
(siRNAs) cause RNA interference
n
encoded by genes in eukaryotic organisms
n
n
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
n
n
Presents a newly identified form of gene regulation
May offer a defense mechanism against certain
viruses
n
n
n
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
n
Random insertion may place an element near a cellular promoter
which will produce a silencing RNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-71
Iron Assimilation and Translation
n
n
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
n
n
It is required for the function of many different enzymes
The assimilation of iron is depicted in Figure 15.26
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-76
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-77
n
Iron is a vital yet potentially toxic substance
n
n
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
n
n
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)
n
n
n
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-78
Figure 15.27 (a) Regulation of ferritin mRNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
15-80