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REGULATION OF GENE
EXPRESSION IN EUKARYOTES
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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INTRODUCTION
• Gene regulation is necessary to ensure
– 1. Expression of genes in an accurate pattern during the
various developmental stages of the life cycle
– Some genes are only expressed during embryonic
stages, whereas others are only expressed in the
adult
– 2. Differences among distinct cell types
– Nerve and muscle cells look so different because of
gene regulation rather than differences in DNA
content
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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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• 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 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
• The sequence it binds is called an enhancer
– A regulatory protein that decreases the rate of
transcription is termed a repressor
• The sequence it binds is called a silencer
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• 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:
• binding of small effector molecules
• protein-protein interactions
• covalent modifications
– Regulatory proteins may alter nucleosomes near the promoter
– DNA methylation may inhibit transcription
• 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
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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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Enhancers and Silencers

Many response elements are orientation
independent or bidirectional
They can function in the forward or reverse orientation
Many response elements are orientation independent or bidirectional. They
can function in the forward or reverse orientation.
5’-GATA-3’
5’-TATC-3’
3’-CTAT-5’
3’-ATAG-5’

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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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
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Coactivator
Activator
TFIID
Enhancer
TFIID
ON
core promoter
Coding sequence
Repressor
OFF
Enhancer
Core
promoter
Coding sequence
Silencer
The activator/coactivator complex recruits TFIID to the core promoter
and/or activates its function. Transcription will be activated.
(a) Transcriptional activation via TFIID
The repressor protein inhibits the binding of TFIID to the core promoter
or inhibits its function. Transcription is repressed.
(b) Transcriptional repression via TFIID
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Core
promoter
ON
Mediator
TFIID
RNA polymerase
and general
transcription
factors
Core
promoter
OFF
Mediator
TFIID
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



The repressor protein interacts with mediator so
that transcription is repressed.
(b) Transcriptional repression via mediator
Transcriptional activator

Transcriptional repressor
stimulates the function of
inhibits the function of
mediator
mediator
This enables RNA pol to form a

Transcription is repressed
preinitiation complex
It then proceeds to the elongation
phase of transcription
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Modulation of Regulatory Transcription Factor
Functions

There are three common ways that the function of
regulatory transcription factors can be affected

1. Binding of a small effector molecule

2. Protein-protein interactions

3. Covalent modification
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The transcription factor
can now bind to DNA
Formation of
homodimers and
heterodimers
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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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ac
5
Lys
Amino-terminal tail
p
ac
p
ac SerLys15
Lys
Ser
ac
Lys
Lys
10
5
20 Lys
10
ac
15
ac
20
H2B
H2A
Globular domain
ac
ac Lys
Lys
10
m ac
Ser
p
m
Arg
Arg Lys
m
ac
Lys
5
m
m
ac
Lys
15
LysSer
10
5
ac
15 Lys
ac
m
Lys
20
Ser
Lys
H4
20
H3
(a) Examples of histone modifications
Core histone
protein
COCH3
Histone
acetyltransferase
COCH3
Histone
deacetylase
COCH3
DNA is less tightly bound
to the histone proteins
Acetyl
group
(b) Effect of acetylation
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A common pattern of nucleosome organization
-2
-1
NFR +1
+3
+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.
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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
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Only one strand is
methylated
Both strands are
methylated

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
 Contain high number of CpG sites
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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
• Methylation may change binding of
transcription factors
• Methyl-CpG-binding proteins may recruit
factors that lead to compaction of the chromatin
• DNA Methylation is Heritable
Transcriptional
activator binds to
unmethylated DNA
This would inhibit the initiation
of transcription
Transcriptional silencing via methylation
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Transcriptional silencing via methylation
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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
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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
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REGULATION OF RNA PROCESSING AND TRANSLATION
• In eukaryotic species, it is common for gene
expression to be regulated at the RNA level
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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

In most cases, large sections of the coding regions are the same
resulting in two alternative versions of a protein that have similar
functions

Nevertheless, there will be enough differences in amino acid
sequences to provide each protein with its own unique characteristics
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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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Alternative Splicing

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
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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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
Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
Alternative Splicing

Alternative splicing is not a random event


The specific pattern of splicing is regulated in a given cell
It involves proteins known as splicing factors

These play a key role in the choice of splice sites
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
The spliceosome recognizes the 5’ and 3’ splice
sites and removes the intervening intron

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

2. Some splicing factors enhance the ability of a
spliceosome to recognize a splice site
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Known as exon
skipping
The role of splicing factors during alternative splicing
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The role of splicing factors during alternative splicing
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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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
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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
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
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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RNA Interference Mediated by Micro RNAs

(miRNAs) 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
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microRNAs can cause RNA degradation and block
translation
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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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
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Protein that carries iron
through the bloodstream
A hollow spherical protein
Prevents toxic buildup of
too much iron in the cell
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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
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(a) Regulation of ferritin mRNA
Ferritin translation is inhibited by low iron, but not by high iron
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(b) Regulation of transferrin receptor mRNA
Increased stability of
mRNA means more
translation
mRNA is degraded and
cannot be translated
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