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Genetics: Analysis and PrinciplesRobert J. Brooker

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 15

GENE REGULATION 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

15-2Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

Figure 15.1 describes the levels of gene expression that are regulated in eukaryotes



Figure 15.1

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


15.1 REGULATORY TRANSCRIPTION FACTORS

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 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

Refer to Figure 15.2

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15-6


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

Structural Features of Regulatory Transcription Factors

15-8


The recognition helix recognizes and makes contact with a base sequence along the major groove of DNA

Hydrogen bonding between an -helix and nucleotide bases is one way a transcription factor can bind to DNA


Composed of one -helix and two -sheets held together by

a zinc (Zn++) metal ionTwo -helices

intertwined due to leucine motifs

Alternating leucine residues in both proteins interact (“zip up”), resulting in protein dimerization

Note: Helix-loop-helix motifs can also mediate protein dimerization

Homodimers are formed by two identical transcription factors;

Heterodimers are formed by two different transcription factors


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

Enhancers and Silencers

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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!

Enhancers and Silencers

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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


TFIID and Mediator

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Figure 15.4


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


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

STOP


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


Regulation of Regulatory Transcription Factors

15-16

Figure 15.5Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15-17

The transcription factor can now bind to DNA

Formation of homodimers and

heterodimers


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

Steroid Hormones and Regulatory Transcription Factors

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Cells respond to steroid hormones in different ways Glucocorticoids

These influence nutrient metabolism in most cells They promote glucose utilization, fat mobilization and protein

breakdown Gonadocorticoids

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

Steroid Hormones and Regulatory Transcription Factors

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15-20Figure 15.6

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



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 cell-signaling 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)

The CREB Protein

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15-22The activity of the CREB proteinFigure 15.7

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

Changes in chromatin structure can involve changes in the structure of DNA and/or changes in chromosomal compaction

These changes include 1. Gene amplification 2. Gene rearrangement 3. DNA methylation 4. Chromatin compaction


15.2 CHANGES IN CHROMATIN STRUCTURE

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Refer to Table 15.1

Uncommon ways to regulate gene expression

Common ways to regulate gene expression


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

Chromatin Structure

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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

15-26

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 -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


Experiment 15A: DNase I Sensitivity and Chromatin Structure

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

Therefore, they had to find a way to specifically monitor the digestion of the -globin gene

They used a radiolabeled cloned DNA fragment (i.e., a probe) that was complementary to the -globin gene

This was hybridized to the chromosomal DNA to determine specifically if the chromosomal -globin gene was intact

Following hybridization, the samples were then exposed to another enzyme, termed S1 nuclease

This enzyme only cuts single-stranded DNA



15-29

Figure 15.9

This indicates that the chromosomal DNA was in a closed conformation

It was inaccessible to DNase I and was thus protected from digestion

This indicates that the chromosomal DNA was in an open conformation

It was accessible to DNase I and was consequently digested

Cut with DNase I Do not cut with DNase I

The Hypothesis

A loosening of chromatin structure occurs when globin genes are transcriptionally active


Testing the Hypothesis


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Figure 15.1015-31

15-32Figure 15.10

15-33Figure 15.10

The Data


Source of nuclei % Hybridization of DNA probe

Reticulocytes 25%

Brain cells >94%

Fibrobalsts >94%

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


The family of globin genes is expressed in the reticulocytes However, individual members are expressed at different

stages of development For example:

-globin Adult -globin Fetus

As shown in Figure 15.11a, several of the globin genes are adjacent to each other on chromosome 11

Globin Gene Expression

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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 -globin even though the gene is perfectly normal

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Figure 15.11

As shown in Figure 15.11b, this type of thalassemia involves a DNA deletion that occurs upstream of the -globin gene

The -globin gene is intact However, it is turned off in these patients

Segments of DNA that are deleted in these populations


A DNA region upstream of the -globin gene was identified as necessary for globin gene expression

This region is termed locus control region (LCR)

It helps in the regulation of chromatin opening and closing

It is missing in certain persons with thalassemias

Figure 15.12

Genes are now accessible to RNA pol and

transcription factors


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


Globin Gene Expression

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15-40

Changes in nucleosome position during the activation of the -globin geneFigure 15.3


Positioned at regular intervals from -3,000 to + 1,500

Disruption in nucleosome positioning from -500 to + 200


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

Chromatin Remodeling

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1. Covalent modification of histones Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation

15-42

Figure 12.13

Adds acetyl groups, thereby loosening the interaction

between histones and DNA

Removes acetyl groups, thereby restoring a tighter interaction


2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of

nucleosomes and thus make the DNA more accessible

15-43

Figure 12.13

Proteins are members of the SWI/SNF family

Acronyms refer to the effects on yeast when these enzyme are

defectiveMutants in SWI are defective in

mating type switching

Mutants in SNF are sucrose non-fermenters

These effects may significantly alter gene expression


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

The gene HO encodes an enzyme that is required for the mating switch


Chromatin Remodeling

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15-45

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

15-46Figure 15.14

SBP is an acronym for a mating type switching cell

cycle box protein)

RNA polymerase


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


DNA Methylation

15-47

15-48Figure 15.15

(or DNA methylase) CH3

CH3

CH3

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

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

15-49

15-50Transcriptional silencing via methylationFigure 15.16

Transcriptional activator binds to

unmethylated DNA

This would inhibit the initiation of transcription

15-51Transcriptional silencing via methylationFigure 15.16


Methylated DNA sequences are inherited during cell division

Figure 15.17 illustrates a model explaining how methylation is passed from mother to daughter cell

DNA Methylation is Heritable

15-52

Figure 15.17

An infrequent and highly regulated event

CH3

CH3

CH3

CH3Hemimethylated

DNA

DNA methylase converts hemi-methylated to

fully- methylated DNA

An efficient and routine event occurring in

vertebrate and plant cells

CH3

CH3CH3CH3

Maintenance methylation

15-53

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


15.3 REGULATION OF RNA PROCESSING AND TRANSLATION

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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

Alternative Splicing

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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


15-57


Figure 15.18 considers an example of alternative splicing for a gene that encodes -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 -tropomyosin by alternative splicing


15-58

15-59Figure 15.18 Alternative ways that the rat -tropomyosin pre-mRNA can be spliced

Found in the mature mRNA from all cell types

Not found in all mature mRNAs

These alternatively spliced versions of -tropomyosin vary in function to meet the needs of the cell type in which they are found


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

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


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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15-62

Figure 15.19 The role of splicing factors during alternative splicing



Figure 15.19 The role of splicing factors during alternative splicing


The term RNA editing refers to a change in the nucleotide sequence of an RNA molecule It involves additions or deletion of particular bases

Or a conversion of one type of base to another

RNA editing can have various effects on mRNAs Generating start or stop codons Changing the coding sequence of a polypeptide

Table 15.3 describes several examples where RNA editing has been found

RNA Editing

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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


RNA Editing

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15-67

3’ end has a sequence of uracils

First, the 5’ anchor binds to target DNA

Cleaves target DNA at a defined location

5’ end is complementary to

mRNA being edited

3’ end of guide RNA becomes displaced from

target DNA

Inserts uracils Removes uracils

Rejoins the two DNA pieces

Figure 15.20


A more widespread mechanism for RNA editing involves changes of one type of base to another This involves deamination of bases

15-68Figure 15.21

Recognized as guanine during

translation


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

Stability of mRNA

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

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 stop codon and the polyA tail

15-71Figure 15.22

3’-untranslated region5’-untranslated region

AU-rich element

Recognized and bound by cellular proteins

These proteins influence mRNA degradation


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

Double-stranded RNA and Gene Silencing

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15-73

Gene insertion leading to the production of double-stranded RNAFigure 15.23Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

PE

PE

PCTranscription occurs from

both promoters

Thus sense and anti-sense strands are transcribed

This produces complementary RNAs that will form a double stranded structure

This event will silence the expression of the cloned gene


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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15-75

Figure 15.24

Cellular mRNA is degraded by endonucleases within the complex

Short RNA from the antisense strand

Thus the expression of the gene that encodes this mRNA is silenced


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


15-76


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

Initiation Factors and the Rate of Translation

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The phosphorylation of initiation factors has been found to affect translation in eukaryotic cells Two initiation factors appear to play a central role in

controlling the initiation of translation eIF2 and eIF4F

The function of these two factors are modulated by phosphorylation in opposite ways Phosphorylation of eIF2 inhibits translation Phosphorylation of eIF4F increases the rate of translation

Figure 15.25 shows the events leading to the translational inhibition by eIF2

15-78

15-79Figure 15.25


Required if eIF2 is to promote binding of the

initiator tRNAmet to the 40S subunit


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

15-80


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

Iron Assimilation and Translation

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15-82Figure 15.26


Protein that carries iron through the bloodstream

A hollow spherical protein

Prevents toxic buildup of too much iron in the cell


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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15-84

Figure 15.27 (a) Regulation of ferritin mRNA

Iron regulatory protein binds IRE

This inhibits translation


Translation of ferritin proceeds

Iron regulatory protein binds iron

It is released from IRE

15-85

Figure 15.27 (b) Regulation of transferrin receptor mRNA


IRP binds IRE

And enhances the stability of mRNA

More mRNA means more translation

IRP binds iron

It is released from IRE

mRNA rapidly degraded

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