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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 15
GENE REGULATION IN
EUKARYOTES
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INTRODUCTION


Eukaryotic organisms have 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 expression
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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 levels of gene expression
that are regulated in eukaryotes
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15-3
Figure 15.1
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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 nearby 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 response elements,
control elements or regulatory elements
The binding of these proteins to these 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
Figure 15.2
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15-7
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 portion of it 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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15-8
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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15-9
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
Note: Helix-loop-helix motifs can
also mediate protein dimerization
Figure 15.3
Alternating leucine residues in
both proteins interact (“zip up”),
resulting in protein dimerization
Homodimers are formed by two
identical transcription factors;
Heterodimers are formed by two
different transcription factors
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15-10
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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15-11
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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15-12
TFIID and Mediator

Most regulatory transcription factors do not bind
directly to RNA polymerase

Two common protein complexes that communicate
the effects of regulatory transcription factors are


1. TFIID

2. Mediator
Refer to Figure 15.4
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15-13
A general transcription factor
that binds to the TATA box
Recruits RNA polymerase
to the core promoter


Transcriptional activator recruits TFIID
to the core promoter and/or activates its
function
Thus, transcription will be activated


Transcriptional repressor inhibits TFIID
binding to the core promoter or inhibits
its function
Thus, transcription will be repressed
Figure 15.4
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15-14
STOP



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


Transcriptional repressor inhibits the
function of mediator
Transcription is repressed
Figure 15.4
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15-14
Regulation of Regulatory
Transcription Factors


There are three common ways that the function of
regulatory transcription factors can be affected

1. Binding of an effector molecule

2. Protein-protein interactions

3. Covalent modification
Refer to Figure 15.5
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15-16
The transcription factor
can now bind to DNA
Formation of
homodimers and
heterodimers
Figure 15.5
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15-17
Steroid Hormones and
Regulatory Transcription Factors

Regulatory transcription factors that respond to
steroid hormones are termed steroid receptors


The hormone actually binds to the factor
The ultimate effect 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
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15-18
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 15.6 shows the stepwise action of a
glucocorticoid hormone
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15-19
Heat shock protein
Heat shock proteins
leave when hormone
binds to receptor
Nuclear localization
Sequence is exposed
Formation of a
homodimer
Glucocorticoid
Response Elements
These function as
enhancers
Transcription of target
gene is activated
Figure 15.6
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15-20
The CREB Protein

The CREB protein is another regulatory
transcriptional factor functioning within living cells


CREB is an acronym for cAMP response element-binding
CREB protein becomes activated in response to cellsignaling molecules that cause an increase in 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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15-21
Could be a hormone,
neurotransmitter,
growth factor, etc.
Acts as a
second
messenger
Activates
protein
kinase A
Phosphorylated CREB
binds to DNA and
stimulates transcription
Unphosphorylated CREB
can bind to DNA, but
cannot activate RNA pol
Figure 15.7
The activity of the CREB protein
15-22
15.2 CHANGES IN
CHROMATIN STRUCTURE

Changes in chromatin structure can involve
changes in the structure of DNA and/or changes in
chromosomal compaction

These changes include

1.
2.
3.
4.

Refer to Table 15.1



Gene amplification
Gene rearrangement
DNA methylation
Chromatin compaction
Uncommon ways to regulate
gene expression
Common ways to regulate
gene expression
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15-23
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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 highly extended
Transcription can take place
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15-25

Variations in the degree of chromatin packing occur
in eukaryotic chromosomes during interphase

During gene activation, tightly packed chromatin must be
converted to an open conformation


In order for transcription to occur
Figure 15.8 shows micrographs of a chromosome
from an amphibian oocyte


The chromosome does not form a uniform 30 nm fiber
Instead many decondensed loops radiate outward


These are DNA regions whose genes are actively transcribed
These chromosomes have been named lampbrush
chromosomes

They resemble brushes once used to clean kerosene lamps
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15-26
Experiment 15A: DNase I
Sensitivity and Chromatin Structure

DNase I is an endonuclease that cleaves DNA


It is much more likely to cleave DNA in an open
conformation than in a closed conformation
In 1976, Harold Weintraub and Mark Groudine used
DNase I sensitivity to study chromatin structure


In particular, they focused attention on the b-globin gene
The gene was known to be specifically expressed in
reticulocytes (immature red blood cells)

But not in other cell types, such as brain cells and fibroblasts
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15-27

First, let’s consider the rationale behind Weintraub
and Groudine’s experimental approach

Globin genes are only a small part of the total DNA


They used a radiolabeled cloned DNA fragment (i.e., a
probe) that was complementary to the b-globin gene


This was hybridized to the chromosomal DNA to determine
specifically if the chromosomal b-globin gene was intact
Following hybridization, the samples were then
exposed to another enzyme, termed S1 nuclease


Therefore, they had to find a way to specifically monitor the
digestion of the b-globin gene
This enzyme only cuts single-stranded DNA
Refer to Figure 15.9
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15-28
Figure 15.9
Cut with DNase I
This indicates that the chromosomal
DNA was in an open conformation
It was accessible to DNase I
and was consequently digested
Do not cut with DNase I
This indicates that the chromosomal
DNA was in a closed conformation
It was inaccessible to DNase I
and was thus protected from digestion
15-29
The Hypothesis

A loosening of chromatin structure occurs when
globin genes are transcriptionally active
Testing the Hypothesis

Refer to Figure 15.10
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15-30
Figure 15.10
15-31
Figure 15.10
15-32
Figure 15.10
15-33
The Data
Source of nuclei
% Hybridization of DNA probe
Reticulocytes
25%
Brain cells
>94%
Fibrobalsts
>94%
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15-34
Interpreting the Data
Source of nuclei

% Hybridization of DNA probe
Reticulocytes
25%
Brain cells
>94%
Fibrobalsts
>94%
Reticulocytes had a much smaller percentage of hybridization

Therefore, their globin genes were more sensitive to DNase I

The globin genes are known to be expressed in reticulocytes but not in
brain cells and fibroblasts

Therefore, these results are consistent with the hypothesis:

The globin gene is less tightly packed when it is being expressed
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15-35
Globin Gene Expression

The family of globin genes is expressed in the
reticulocytes


However, individual members are expressed at different
stages of development
For example:



b-globin  Adult
g-globin  Fetus
As shown in Figure 15.11a, several of the globin
genes are adjacent to each other on chromosome 11
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15-36
Figure 15.11
Segments of DNA that are deleted in these populations

Thalassemia is a defect in the expression of one or more
globin genes


An intriguing observation of some thalassemic patients is that they
cannot synthesize b-globin even though the gene is perfectly normal
As shown in Figure 15.11b, this type of thalassemia involves a
DNA deletion that occurs upstream of the b-globin gene


The b-globin gene is intact
However, it is turned off in these patients
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15-37


A DNA region upstream
of the b-globin gene
was identified as
necessary for globin
gene expression
This region is termed
locus control region
(LCR)


Genes are now accessible
to RNA pol and transcription
factors
It helps in the regulation
of chromatin opening
and closing
It is missing in certain
persons with
thalassemias
Figure 15.12
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15-38
Globin Gene Expression

Aside from chromatin packing, a second structural
issue to consider is the position of nucleosomes

In chromatin, the nucleosomes are usually
positioned at regular intervals along the DNA

However, they have been shown to change positions in
cells that normally express a particular gene


But not in cells where the gene is inactive
Refer to Figure 15.13
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15-39
Positioned at regular intervals from -3,000 to + 1,500
Disruption in nucleosome positioning
from -500 to + 200
Figure 15.3
Changes in nucleosome position during the activation of the b-globin gene
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15-40
Chromatin Remodeling


As discussed in Chapter 12, there are two common
ways in which chromatin structure is altered

1. Covalent modification of histones

2. ATP-dependent chromatin remodeling
So let’s review Figure 12.13
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15-41

1. Covalent modification of histones

Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation
Adds acetyl groups, thereby
loosening the interaction
between histones and DNA
Figure 12.13
Removes acetyl groups,
thereby restoring a
tighter interaction
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15-42

2. ATP-dependent chromatin remodeling

The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
Proteins are members of the
SWI/SNF family
Acronyms refer to the effects on yeast
when these enzyme are defective
Mutants in SWI are defective in
mating type switching
Mutants in SNF are
sucrose non-fermenters
Figure 12.13
These effects may significantly alter
gene expression
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15-43
Chromatin Remodeling

An important role for transcriptional activators is to
recruit the aforementioned enzymes to the promoter

A well-studied example of recruitment involves a
gene in yeast that is involved in mating



Yeast can exist in two mating types, termed a and a
The gene HO encodes an enzyme that is required for the
mating switch
Refer to Figure 15.14
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15-44
SWI refers to mating type
switching
SAGA is an acronym for
Spt/Ada/GCN5/Acetyltransferase
Genes known to be transcriptionally
regulated by histone acetyltransferase
Figure 15.14
15-45
SBP is an acronym for a
mating type switching cell
cycle box protein)
Figure 15.14
RNA polymerase
15-46
DNA Methylation

DNA methylation is a change in chromatin structure
that silences gene expression

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 15.15
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15-47
(or DNA methylase)
CH3
Only one strand is
methylated
CH3
Both strands are
methylated
CH3
Figure 15.15
15-48

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



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
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15-49
Transcriptional
activator binds to
unmethylated DNA
This would inhibit the
initiation of transcription
Figure 15.16 Transcriptional silencing via methylation
15-50
Figure 15.16 Transcriptional silencing via methylation
15-51
DNA Methylation is Heritable

Methylated DNA sequences are inherited during
cell division

Figure 15.17 illustrates a model explaining how
methylation is passed from mother to daughter cell
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15-52
Figure 15.17
An infrequent and
highly regulated event
CH3
CH3
Hemimethylated
DNA
CH3
CH3
Maintenance methylation
CH3
CH3
DNA methylase converts
hemi-methylated to
fully- methylated DNA
An efficient and routine
event occurring in
vertebrate and plant cells
CH3
CH3
15-53
15.3 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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15-54
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15-55
Alternative Splicing

One very important biological advantage of introns in
eukaryotes is the phenomenon of alternative splicing

Alternative splicing refers to the fact that pre-mRNA
can be spliced in more than one way

In most cases, this produces two alternative versions of a
protein that have similar functions


Because much of their amino acid sequences are identical
Nevertheless, there will be enough differences in amino
acid sequences to provide each protein with its own
characteristics
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15-56
Alternative Splicing

The degree of splicing and alternative splicing
varies greatly among different species

Baker’s yeast contains about 6,000 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 ~ 35,000 genes

Most of these encode mRNAs that are spliced


It is estimated that a minimum of one-third of are alternatively spliced
Note: Certain mRNAs can be alternatively spliced to produce dozens
or even hundreds of different mRNAs
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15-57
Alternative Splicing

Figure 15.18 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)
The different cells of a multicellular organism regulate
their contraction in subtly different ways

One way to accomplish this is to produce different forms of
a-tropomyosin by alternative splicing
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15-58
Found in the mature mRNA
from all cell types
Not found in all
mature mRNAs
These alternatively spliced versions of a-tropomyosin vary in
function to meet the needs of the cell type in which they are found
Figure 15.18
Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
15-59
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 is 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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15-60

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 15.19a
2. Some splicing factors enhance the ability of a
spliceosome to recognize a splice site

Refer to Figure 15.19b
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15-61
Figure 15.19 The role of splicing factors during alternative splicing
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15-62
Figure 15.19 The role of splicing factors during alternative splicing
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15-63
RNA Editing

The term RNA editing refers to a change in the
nucleotide sequence of an RNA molecule

It involves additions or deletion of particular bases


RNA editing can have various effects on mRNAs



Or a conversion of one type of base to another
Generating start or stop codons
Changing the coding sequence of a polypeptide
Table 15.3 describes several examples where RNA
editing has been found
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15-64
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15-65
RNA Editing

RNA editing was first discovered in trypanosomes

The protozoa that cause sleeping sickness

In these organisms, the process involves guide RNA

Guide RNA can direct the addition or deletion of one
or more uracils into an RNA

Refer to Figure 15.20
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15-66
5’ end is
complementary to
mRNA being edited
3’ end has a
sequence of uracils
Cleaves target DNA at a
defined location
First, the 5’ anchor
binds to target DNA
3’ end of guide RNA
becomes displaced from
target DNA
Removes uracils
Inserts uracils
Rejoins the two DNA pieces
Figure 15.20
15-67

A more widespread mechanism for RNA editing
involves changes of one type of base to another

This involves deamination of bases
Recognized as
guanine during
translation
Figure 15.21
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15-68
Stability of mRNA

The stability of eukaryotic mRNA varies considerably


Several minutes to several days
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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15-69

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 nucleases
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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15-70

2. Destabilizing elements


Found especially in mRNAs that have short half-lives
These elements can be found anywhere on the mRNA

However, they are most common at the 3’ end between the
codon and the polyA tail
stop
AU-rich element
Recognized and bound by cellular proteins
These proteins influence mRNA degradation
5’-untranslated region
Figure 15.22
3’-untranslated region
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15-71
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
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15-72
PE
This event will silence the
expression of the cloned gene
PE
PC
Transcription occurs from both
promoters
Thus sense and anti-sense
strands are transcribed
This produces complementary
RNAs that will form a
double stranded structure
Figure 15.23 Gene insertion leading to the production of double-stranded RNA
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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

This phenomenon was termed RNA interference (RNAi)

A proposed mechanism for RNAi is shown in Figure 15.24
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15-74
Short RNA from the
antisense strand
Thus the expression of
the gene that encodes
this mRNA is silenced
Cellular mRNA is degraded by
endonucleases within the complex
Figure 15.24
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Double-stranded RNA and Gene
Silencing

RNA interference is widely found in eukaryotes

It is believed to

1. Offer a host defense mechanism against certain viruses


Those with double-stranded RNA genomes, in particular
2. Play a role in silencing certain transposable elements

Some of these elements produce double-stranded RNA
intermediates as part of the transposition process
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15-76
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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15-77

The phosphorylation of initiation factors has been
found to affect translation in eukaryotic cells


The function 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 the
translational inhibition by eIF2a
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15-78
Required if eIF2 is to
promote binding of the
initiator tRNAmet to the 40S
subunit
Figure 15.25
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15-79

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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15-80
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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15-81
Protein that carries iron
through the bloodstream
A hollow spherical protein
Prevents toxic buildup of
too much iron in the cell
Figure 15.26
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
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)


IRE is found in the 5’-UTR in ferritin mRNA
 And in the 3’-UTR in transferrin receptor mRNA
Regulation of iron assimilation is shown in Figure 15.27
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Figure 15.27 (a) Regulation of ferritin mRNA
Iron regulatory protein
binds IRE
This inhibits translation
Iron regulatory protein
binds iron
It is released from IRE
Translation of ferritin
proceeds
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15-84
Figure 15.27 (b) Regulation of transferrin receptor mRNA
More mRNA
means more
translation
IRP binds IRE
And enhances the
stability of mRNA
IRP binds iron
It is released from IRE
mRNA rapidly degraded
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