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Chapter 23
The Regulation
of Gene
Expression
Lectures by
Kathleen Fitzpatrick
Simon Fraser University
© 2012 Pearson Education, Inc.
The Regulation of Gene Expression
• Regulation is an important aspect of almost every
process in nature, especially gene expression
• Selective gene expression allows cells to be
efficient, synthesizing only what is needed for each
cell type
• The first understanding of gene regulation came
from bacteria
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Bacterial Gene Regulation
• Some genes are constitutive, expressed all the time
• For most others, expression is regulated to meet the
cell’s needs
• Regulated genes encode enzymes needed for
processes that are not constantly required
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Catabolic and Anabolic Pathways Are
Regulated Through Induction and
Repression, Respectively
• Bacteria use different approaches for regulating
enzyme synthesis depending on whether an
enzyme is involved in a catabolic (degradative) or
anabolic (synthetic) pathway
• Enzymes that catalyze such pathways are often
regulated coordinately
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Catabolic Pathways and Substrate
Induction
• Catabolic enzymes exist for the purpose of
degrading specific substrates
• In the catabolic pathway that degrades lactose, the
central step in the pathway is catalyzed by
-galactosidase
• Before lactose can be hydrolyzed, it must be
transported into the cell via a protein called
galactosidase permease
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Substrate induction
• -galactosidase is needed only when lactose is
present
• Therefore, it is turned on, induced, only in the
presence of lactose, an example of substrate
induction
• Enzymes whose synthesis is regulated this way are
called inducible enzymes
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Anabolic Pathways and End-Product
Repression
• For anabolic pathways, the amount of enzyme
produced by a cell usually correlates inversely with
the concentration of the end product of the pathway
• E.g., as the concentration of tryptophan rises, it is
efficient for the cell to reduce the production of the
enzymes needed for tryptophan synthesis
• This is end-product repression
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Repression and inhibition
• Repression is a general term, referring to reduced
expression of any regulated gene
• Genetic repression has an effect on protein
synthesis, not just protein activity
• In feedback inhibition molecules of enzyme are still
present but their catalytic activity is inhibited
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Effector Molecules
• In both induction and repression of enzyme
synthesis, control is triggered by small organic
molecules, present in the cell or its surroundings
• The small organic molecules are called effectors
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The Genes Involved in Lactose
Catabolism Are Organized into an
Inducible Operon
• The cornerstone of the lactose operon model rests
on the discovery of two types of genes
• The first are the genes coding for enzymes used
for lactose uptake and metabolism
• The second is a regulatory gene whose product
controls activity of the first group
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Genes for lactose metabolism
• The lacZ gene encodes -galactosidase, which
hydrolyzes lactose
• The lacY gene encodes galactosidase permease,
involved with uptake of lactose from outside the cell
• The lacA gene encodes a transacetylase that adds
an acetyl group to lactose as it enters the cell
© 2012 Pearson Education, Inc.
The operon
• The three lac genes belong to a single regulatory
unit, an operon
• An operon is a group of genes with related function
that are clustered together and are turned on and
off simultaneously
• This is less common in eukaryotes than in
prokaryotes
© 2012 Pearson Education, Inc.
The lac Operon Is Negatively
Regulated by the lac Repressor
• In an operon, genes with related functions are
clustered together so their transcription can be
coordinately regulated
• In order for inducers such as lactose to turn on the
genes, an additional gene is needed, the lacI gene
• In the absence of the lacI gene, cells always
produce the lacZ, lacY, and lacA gene products,
even when lactose is not present
© 2012 Pearson Education, Inc.
The lac operon
• The lacI gene codes for a product that regulates
expression of the lacZ, lacY, and lacA genes
• A regulatory gene product that inhibits the
expression of other genes is called a repressor
protein
• The lac operon consists of the three above genes
preceded by a promoter (Plac) and a sequence
called the operator (O)
© 2012 Pearson Education, Inc.
Transcription of the lac operon
• Transcription of the operon begins at the promoter
and proceeds through the operator and the lacZ,
lacY, and lacA genes
• The result is one transcript that codes for all three
gene products
• mRNAs that code for more than one polypeptide
are called polycistronic mRNAs
© 2012 Pearson Education, Inc.
Figure 23-2
© 2012 Pearson Education, Inc.
Control of the lac operon
• The interaction between the operator site and the
repressor protein is crucial to lac operon control
• The lacI gene encodes the lac repressor, and is
located just outside the operon
• The repressor binds the operator, preventing RNA
polymerase from transcribing the lac genes
© 2012 Pearson Education, Inc.
Figure 23-3A
© 2012 Pearson Education, Inc.
Regulation of the lac operon
• Inducer molecules bind to the repressor, altering its
conformation so that it cannot bind the operator any
longer
• Once the operator is no longer blocked by the
repressor, the RNA polymerase can transcribe the
lacZ, lacY, and lacA genes
• The repressor is an allosteric protein, with two
possible conformational states
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Figure 23-3B
© 2012 Pearson Education, Inc.
Allolactose is the inducer
• The effector for the repressor is allolactose, an
isomer of lactose produced after lactose enters
the cell
• The binding of allolactose to the repressor induces a
conformational change that renders the repressor
incapable of binding the operator
• In this way lactose triggers the induction of the lac
operon
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Studying the lac operon
• To study the lac operon, a synthetic inducer,
-galactoside isopropylthiogalactoside (IPTG) is
used as the inducing molecule
• The term inducer is used to describe any effector
that turns on transcription of an inducible operon
• The lac operon is an inducible operon because it
is turned off unless induced
© 2012 Pearson Education, Inc.
Studies of Mutant Bacteria Revealed
How the lac Operon Is Organized
• Much of the early evidence for the operon model
involved studies of mutant bacteria
• Some mutations were located in the operon genes,
which affect only one of the protein products
• Others occur in the regulatory elements of the
operon that affect all of the genes coordinately
© 2012 Pearson Education, Inc.
Table 23-1
© 2012 Pearson Education, Inc.
Operon Gene Mutations
• Mutations in the lacY or lacZ genes lead to
production of defective enzymes even in the
presence of inducer
• The mutants are unable to use lactose as a carbon
source
• A defective gene or regulatory sequence is
indicated by a superscript minus sign, e.g., Y
© 2012 Pearson Education, Inc.
Operator Mutations
• Mutations in the operator can lead to constitutive
expression
• In these, the mutant cells continue to produce the
lac enzymes whether inducer is present or not
• Operator-constitutive mutants are represented
as Oc
© 2012 Pearson Education, Inc.
Promoter Mutations
• Promoter mutations can decrease the affinity of
RNA polymerase for the promoter so that the rate of
mRNA production decreases
• P mutations decrease both the elevated rate of
enzymes produced in presence of inducer, and the
low, basal level of production without inducer
© 2012 Pearson Education, Inc.
Regulatory Gene Mutations
• There are two types of mutations in the lacI gene
• Some mutants fail to produce any of the lac
enzymes whether or not inducer is present
• These are called superrepressor mutants (Is)
• The repressor remains bound to the operator
whether or not inducer is present
© 2012 Pearson Education, Inc.
Regulatory gene mutations (continued)
• The other class of lacI mutations produces a
repressor protein that does not recognize the
operator (or is not synthesized at all)
• The lac operon in such I mutants cannot be turned
off, so that the enzymes are synthesized
continuously
© 2012 Pearson Education, Inc.
The Cis-Trans Test Using Partially
Diploid Bacteria
• A cis-trans test is used to differentiate between
cis-acting mutations, which affect DNA sites, and
trans-acting mutations, which affect proteins
• Partially diploid bacteria can be constructed by
inserting a second copy of the lac portion of the
bacterial chromosome into the F-factor of F+ cells
© 2012 Pearson Education, Inc.
Table 23-2
© 2012 Pearson Education, Inc.
The cis-trans test
• If only one copy of the operon contains an I or Oc
mutation, it is possible to determine if the mutation
affects both copies of the operon
• A partial diploid can be made with one copy of the
operon containing a Z mutation and the other copy
the Y mutation
• In this way, the effect of regulatory mutations can be
studied
© 2012 Pearson Education, Inc.
The cis-trans test
• Mutations that affect only the genes to which they
are physically connected are said to be cis-acting
• Mutations that can affect both copies of the operon
are said to be trans-acting
• E.g., I+ allows for proper gene expression even in
the presence of an I mutation; thus the repressor is
called a trans-acting factor
© 2012 Pearson Education, Inc.
O mutations are cis-acting
• In contrast to the I gene, O locus mutants act in cis
• E.g., Oc affects only genes physically connected to
it; thus the O site is a cis-acting element
• Cis-specificity is characteristic of mutations that
affect binding sites on DNA rather than protein
products
© 2012 Pearson Education, Inc.
Catabolite Activator Protein (CAP)
Positively Regulates the lac Operon
• Glucose is the preferred carbon source for almost
all cells
• Catabolite repression is used by bacteria to ensure
that glucose is metabolized preferentially when
available
• It is an example of positive transcriptional control
(as opposed to negative control of lac operon
regulation)
© 2012 Pearson Education, Inc.
The effector molecule is cAMP
• In catabolite repression, the effector molecule is
cyclic AMP (cAMP)
• Glucose inhibits adenylyl cyclase; the more
glucose is present, the less cAMP is made
• Catabolite activator protein (CAP) is an activator
protein that turns on transcription and can bind
DNA of some operons when it is complexed
with cAMP
© 2012 Pearson Education, Inc.
CAP regulation of transcription
• The CAP-binding site is located upstream of the
promoter in the lac operon
• Similar sites are found in the inducible operons for
metabolism of other sugars
• The binding of activated (cAMP-bound) CAP
greatly enhances binding of RNA polymerase to
the promoter, promoting operon transcription when
glucose is absent
© 2012 Pearson Education, Inc.
Figure 23-4, Steps 1, 2
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Figure 23-4, Steps 3, 4
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The lac Operon Is an Example of the
Dual Control of Gene Expression
• The lac operon is subject to both negative and
positive control
• Most tightly regulated genes in eukaryotes and
prokaryotes are under such dual control
• Factors whose binding of DNA inhibit transcription
are negative regulators; those that enhance
transcription are positive regulators
© 2012 Pearson Education, Inc.
The Structure of the lac
Repressor/Operator Complex Confirms
the Operon Model
• In 1996, Lewis, Lu, and colleagues reported the
structure of the lac repressor protein bound to the
operator site
• This confirmed genetic and biochemical studies
• The repressor interacts with the operator as a
tetramer
© 2012 Pearson Education, Inc.
The repressor/operator complex
• The primary operator (O1) is 21 bp long
• There are two additional sequences called O2, O3;
when repressor binds O1 and O3 a loop is formed
that prevents RNA polymerase from moving along
the operon
• When allolactose binds the repressor, it releases
the DNA, which relaxes and allows transcription
© 2012 Pearson Education, Inc.
Figure 23-5
© 2012 Pearson Education, Inc.
Figure 23-5A
© 2012 Pearson Education, Inc.
Figure 23-5B
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Figure 23-5C
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The Genes Involved in Tryptophan
Synthesis Are Organized into a
Repressible Operon
• Operons coding for enzymes involved in catabolic
pathways generally resemble the lac operon in
being inducible
• Operons that regulate enzymes involved in
anabolic pathways are repressible operons
• They are turned off allosterically, usually by an
effector that is the end-product of the pathway
© 2012 Pearson Education, Inc.
The trp operon
• The trp operon contains genes coding for enzymes
needed in tryptophan biosynthesis and the
accompanying regulatory DNA sequences
• The effector molecule is the end-product of the
pathway, tryptophan
• Production of trp biosynthetic enzymes is
repressed in the presence of trp
© 2012 Pearson Education, Inc.
The trp repressor
• The regulatory gene for the trp operon is called
trpR; it encodes a repressor protein that is active
when bound to the effector
• Tryptophan can be considered a corepressor,
because it is required, along with the repressor
protein, to shut off expression of the operon
© 2012 Pearson Education, Inc.
Figure 23-6A
© 2012 Pearson Education, Inc.
Figure 23-6B
© 2012 Pearson Education, Inc.
Sigma Factors Determine Which
Sets of Genes Can Be Expressed
• Bacterial cells employ different sigma (s) factors to
control which genes will be transcribed
• s factors recognize gene promoters; the most
common one in E. coli is s70, though there are
others used when environmental changes occur
• Alternative s factors alter promoter recognition,
allowing cells to adapt to the changed conditions
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Sigma factors
• Different types of bacteria have different numbers
of alternative sigma factors
• Some are produced by bacteriophages, which
allows the phage to take over the transcriptional
machinery of the cell
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Attenuation Allows Transcription to Be
Regulated After the Initiation Step
• Bacteria employ additional regulatory mechanisms
that operate after transcription is initiated
• The trp operon of E. coli has an unusual leader
sequence between the promoter and the first gene,
trp E.
• The leader sequence is transcribed even in the
absence of trp
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Attenuation in the trp operon
• A portion of the leader sequence is translated,
forming a leader peptide 14 amino acids long
• The leader sequence mRNA contains two
adjacent codons for trp
• The leader also has 4 regions (numbered 1–4)
that can pair to form distinctive hairpin loops
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Figure 23-7
© 2012 Pearson Education, Inc.
Attenuation in the trp operon (continued)
• Regions 3 and 4 of the leader mRNA plus eight
adjacent Us comprise the terminator
• When trp levels are low, a ribsome that reaches
the trp codons will pause, awaiting the tRNA trp
• The stalled ribosome blocks region I, promoting a
hairpin structure to form between regions 2 and 3
(the antiterminator hairpin)
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Attenuation in the trp operon (continued)
• When region 3 is paired with region 2, it cannot
form the termination structure with region 4, so
transcription continues
• However, if trp is plentiful, the ribosome does not
pause and continues to the stop codon in region 2
• This permits interaction between regions 3 and 4,
which terminates transcription
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Figure 23-8A
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Figure 23-8B
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Figure 23-8C
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Attenuation is fairly common
• Although attenuation was once considered to be an
unusual type of regulation, it is relatively common
• This is especially true for operons involved in amino
acid biosynthesis
• In some operons, attenuation complements
regulation via the operator; in others it is the only
means of regulation
© 2012 Pearson Education, Inc.
Riboswitches Allow Transcription and
Translation to Be Controlled by Small
Molecule Interactions with RNA
• Small molecules can regulate gene expression by
binding to special sites in mRNA called
riboswitches
• Binding of the molecule to its riboswitch triggers
changes in mRNA shape that affect transcription or
translation
• Riboswitches are typically found in the untranslated
leader region of mRNAs of bacterial operons
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One of the first riboswitches to be
discovered
• One of the first riboswitches identified is RNA from
the riboflavin (rib) operon of the bacterium B. subtilis
• The RNA of the rib operon possesses a leader
sequence that can fold into a hairpin loop that
terminates transcription
• The coenzymes FMN and FAD are end-products of
the operon; binding of FMN to the leader sequences
promotes formation of a termination hairpin
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Figure 23-9A
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Riboswitches can control translation
• The binding of small molecules to riboswitches can
also control translation
• The genes involved in synthesizing FMN and FAD in
E. coli are not clustered in an operon but some are
controlled by riboswitches
• Binding of RMN to its riboswitch promotes formation
of a hairpin that includes sequences required for
binding the mRNA to ribosomes
© 2012 Pearson Education, Inc.
Figure 23-9B
© 2012 Pearson Education, Inc.
Eukaryotic Gene Regulation: Genomic
Control
• In some fundamental features, there are numerous
similarities between bacteria and eukaryotes
• However, eukaryotes require a diversity of genetic
control mechanisms, some of which are quite
different from those of bacteria
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Multicellular Eukaryotes Are Composed
of Numerous Specialized Cell Types
• In the case of multicellular eukaryotes, a single
organism consists of a mixture of specialized or
differentiated cell types
• These are distinguished from each other based on
difference in appearance and protein products
• Such differences indicate that differential gene
expression plays a central role in creating
differentiated cells
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Differentiation
• Differentiated cells are produced from groups of
immature, nonspecialized cells
• The process is called cell differentiation
• The classic example occurs in embryos, in which
cells of the early embryo produce all the cell types
that make up the organism
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Eukaryotic Gene Expression Is
Regulated at Five Main Levels
• The overall pattern of gene expression results from
controls exerted at several different levels
• Control is exerted at five levels: the genome (1),
transcription (2), RNA processing and export (3),
translation (4), and posttranslational events (5)
• The last three categories represent levels of
posttranscriptional control
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Figure 23-10, Parts 1–3
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Figure 23-10, Parts 4, 5
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As a General Rule, the Cells of a
Multicellular Organism All Contain the
Same Set of Genes
• The first level of control is exerted at the level of the
overall genome
• Though each specialized cell type uses only a
fraction of the genes in the genome, almost all cells
retain the same complete set of genes
• In 1958, this was demonstrated by Steward, who
grew entire carrot plants from single root cells
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More evidence from the African clawed
frog (Xenopus laevis)
• In 1964, Gurdon and colleagues transplanted nuclei
from differentiated tadpole cells into unfertilized
eggs deprived of their own nuclei
• The frequency of success was low, but some of the
eggs developed into swimming tadpoles
• These tadpoles, created by nuclear transplantation,
were clones of the individual from which the original
nucleus was taken
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Cloning mammals
• In 1997 Wilmut and colleagues cloned a sheep,
Dolly, the first mammal cloned from a cell that came
from an adult
• Similar techniques have been used to clone other
mammals
• Nuclei that contain a complete set of genes, and
can direct formation of a new organism, are said to
be totipotent
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Figure 23A-1A, Steps 1, 2
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Figure 23A-1A, Steps 3, 4
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Figure 23A-1A, Steps 5, 6
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Figure 23A-1B
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Gene Amplification and Deletion Can
Alter the Genome
• A few types of gene regulation create exceptions to
the rule that the genome tends to be identical in all
the cells of an adult eukaryote
• Gene amplification is used to make multiple copies
of the same gene
• This is an example of genomic control, a
regulatory change in the makeup or organization of
a genome
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Amplification of rRNA genes in
Xenopus laevis
• The haploid genome of Xenopus has about 500
copies of genes for 5.8S, 18S, and 28S rRNA
• However, the genes are selectively replicated about
4000-fold during oogenesis so that the mature
oocyte contains about 2 million copies
• This amplification is needed to produce enough
ribosomes during oogenesis, enough to sustain the
embryo through its early development
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Gene deletion also occurs
• Some cells delete genes whose products are not
required
• Extreme gene deletion (DNA diminution) occurs in
mammalian red blood cells
• These discard their nuclei once sufficient
hemoglobin RNA is synthesized
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DNA Rearrangements Can Alter the
Genome
• In a few cases, gene regulation is based on the
movements of DNA segments from one location
to another within the genome
• This is called DNA rearrangement
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Yeast Mating-Type Rearrangements
• In yeast, Saccharomyces cerevisiae mating occurs
when haploid cells of different mating types (a and
) fuse to form a diploid cell
• Cells have alleles for both mating types, but the
actual mating type depends on which of the alleles
is present at a special site in the genome called the
MAT locus
• Cells switch mating type by moving the alternate
allele into the MAT locus
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The cassette mechanism
• The process of DNA rearrangement involved in
mating type switching is called the cassette
mechanism
• The MAT locus containing the a or  allele is on
chromosome 3, between extra copies of the two
alleles
• The extra copies are called HML and HMRa
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Mechanism of switching
• To switch mating type, the HO endonuclease
creates a break in the chromosome at the MAT
locus
• Endonucleases degrade the cut DNA
• HML or HMRa DNA is used as a template to
repair the gap in the DNA, which results in a switch
of the mating type allele found at the MAT locus
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No expression from the HML and
HMR genes
• Though the yeast cells contain genes for both
mating type proteins, only the one from the MAT
locus is expressed
• A group of regulatory proteins, the silent
information regulator (SIR) genes, prevent
expression at HML and HMR
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Figure 23-11
© 2012 Pearson Education, Inc.
Antibody Gene Rearrangements
• Lymphocytes of the vertebrate immune system
undergo DNA rearrangement to produce antibodies
• Antibodies are composed of heavy chains and light
chains
• Millions of different kinds of antibodies can be made
by the vertebrate immune system
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Antibody production
• Lymphocytes use a relatively small number of DNA
segments and rearrange them in various
combinations to produce millions of unique antibody
genes
• Four kinds of DNA segments are involved: V, J, D,
and C segments
• The C segment encodes constant regions of the
heavy or light chains, the others are variable regions
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Antibody production (continued)
• Heavy chains are constructed from about 200 kinds
of V segments, more than 20 kinds of D segments,
and at least 6 kinds of J segments
• These are rearranged during lymphocyte
development, when one of each type of segment is
brought together at random, to generate at least
24,000 different kinds of heavy chain variable
regions
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Antibody production (continued)
• Light chains are similarly variable, though they do
not use D segments
• Further variation is produced as any light chain is
assembled with any type of heavy chain to create
millions of different combinations
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Figure 23-12
© 2012 Pearson Education, Inc.
Chromosome Puffs Provide Visual
Evidence That Chromatin
Decondensation Is Involved in
Genomic Control
• In eukaryotes, some degree of chromatin
decondensation is necessary for gene expression
• Evidence of decondensation came from microscopic
visualization of certain insect chromosomes
• In the fruit fly, tissues such as the salivary glands
contain large polytene chromosomes
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Polytene chromosomes
• Polytene chromosomes form by successive
replication without subsequent division
• The newly formed chromatids line up in parallel to
form the multistranded polytene chromosomes
• These large chromosomes have a characteristic
banding pattern and are easily visualized
microscopically
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Chromosome puffs
• Transcriptional activation of genes in a particular
chromosome band causes the compacted
chromatin to uncoil and expand
• A visible chromosome puff is formed
• Puffs are not the only sites of transcription, but the
extent of decondensation at puff sites correlates
well with the level of transcription
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Chromosome puffs and ecdysone
• As the fruit fly (Drosophila) larvae progress through
various stages, the pattern of chromosome puffing
changes
• The puffs respond to the steroid hormone, ecdysone
• It binds to and activates a regulatory protein that
stimulates expression of certain genes at certain
times
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Figure 23-13
© 2012 Pearson Education, Inc.
DNAse I Sensitivity Provides Further
Evidence for the Role of Chromatin
Decondensation in Genomic Control
• DNAse I is an endonuclease that preferentially
degrades transcriptionally active DNA in chromatin
• The increased sensitivity of such regions to
digestion suggests that their DNA is uncoiled
• E.g., the globin gene is sensitive to low levels of
DNAse I in chicken red blood cells (erythrocytes),
where it is expressed, but not in other cells
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Figure 23-14
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DNAse I hypersensitivity
• When nuclei are treated with very low levels of
DNAse I, specific regions that are very susceptible
to digestion can be detected
• These DNAse I hypersensitive sites tend to occur
up to a few hundred bases upstream from the
transcription start sites of active genes
• These sites correspond to regions where DNA is
nucleosome free
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Figure 23-15
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DNA Methylation Is Associated with
Inactive Regions of the Genome
• DNA methylation, the addition of methyl groups to
certain cytosine bases in DNA, can reduce gene
activity
• Methylation of promoter regions can block access of
proteins needed for transcription, or serve as binding
sites for proteins that condense chromatin
• The net effect is gene silencing
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DNA methylation
• DNA methylations occur on cytosines in 5-CG-3
sequences base-paired to 3-GC-5 sequences that
are already methylated
• This allows methylation patterns on DNA to be
inherited
• This can create epigenetic changes, stable
alterations in gene expression, transmitted from one
cellular generation to the next, with no change in
DNA sequence
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X-inactivation
• Female mammals have two X chromosomes,
whereas males have only one
• One of the X chromosomes in females is randomly
inactivated during early embryonic development
• In X-inactivation, the DNA of one X is extensively
methylated and the chromatin condenses into a tight
mass of heterochromatin, inaccessible to
transcription factors
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X-inactivation
• The inactivated X chromosome is visible as a dark
spot called a Barr body
• Once a certain X is inactivated in a cell, all the
daughter cells produced by the original have the
same X inactivated
• The result of X-inactivation is that females and
males each have one active X chromosome per
adult cell
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Methylation and restriction enzymes
• Evidence of connection of DNA methylation and
gene inhibition comes from studies using the
restriction enzymes HpaII and MspI
• Both enzymes cleave the recognition site -CCGGbut HpaII cuts only if the central C is unmethylated
• Comparing fragments generated by digesting with
both enzymes shows that DNA sites are methylated
in a tissue-specific fashion
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Figure 23-16
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Figure 23-16A
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Figure 23-16B
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Imprinting and methylation
• Genomic imprinting causes certain genes to be
expressed differently depending on the parent from
which they are inherited
• This differing behavior results from differential
methylation
• Some genes are maternally imprinted (methylated)
so only the paternal copy is active; and vice versa
for paternally imprinted genes
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Imprinting syndromes
• Prader-Willi syndrome is characterized by deletion
of paternal genes (the maternal copies of which
are methylated) from a particular region of
chromosome 15
• Angelman syndrome results when a gene in this
region is deleted from the maternal chromosome;
the paternal copy of the gene is inactive
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Changes in Histones and Chromatin
Remodeling Proteins Can Alter
Genome Activity
• Histone proteins affect packaging of DNA; their
protruding tails can be tagged by addition of
phosphate, methyl, or acetyl group
• Combinations of these tags create a histone code,
a set of signals for modifying chromatin structure
and gene activity
• Methylation of lysine can serve as a signal for
activation or repression of gene activity
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Histone methylation and acetylation
• Methylation of lysine 4 of histone H3 is a mark of
active genes, whereas methylation of lysine 9 and
27 is associated with gene silencing
• Acetylation is accomplished by the enzyme histone
acetyl transferase (HAT) and generally promotes
chromatin decondensation
• Histone deacetylase (HDAC) removes acetyl
groups from histones
© 2012 Pearson Education, Inc.
Figure 23-17A
© 2012 Pearson Education, Inc.
Gene activation and repression
• Repressor proteins which cause transcription to
occur less frequently, recruit HDAC complexes to
deacetylate histones
• Activator proteins recruit HAT complexes
• DNAse I digestion releases acetylated histones H3
and H4, suggesting that they are preferentially
associated with active genes
© 2012 Pearson Education, Inc.
Histone H1 and HMG proteins
• Studies show that transcriptionally active chromatin
often lacks histone H1
• Histone H1 is required for folding chromatin into 30
nm fibers, suggesting that its absence helps
maintain active chromatin in the 10 nm form
• Active chromatin also has a large content of HMG
(high-mobility group) proteins, non-histone proteins
that bind selectively to active genes
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Chromatin remodeling
• In addition to modifying histones, other proteins
alter the position of nucleosomes along the DNA
• These chromatin remodeling proteins use the
energy of ATP hydrolysis to slide nucleosomes or
cause their ejection from a region of chromatin
• One important class of these proteins is the
SWI/SNF family
© 2012 Pearson Education, Inc.
Figure 23-17B
© 2012 Pearson Education, Inc.
Eukaryotic Gene Regulation:
Transcriptional Control
• Transcriptional control is the second main
level for controlling gene expression
© 2012 Pearson Education, Inc.
Different Sets of Genes Are
Transcribed in Different Cell Types
• Different proteins produced by two cell types in the
same individual result from differential gene
transcription
• Nuclear run-on transcription experiments provide a
shapshot of the transcription in a nucleus at a given
moment
• These experiments demonstrate that transcribed
RNAs in different cell types are different
© 2012 Pearson Education, Inc.
Figure 23-18
© 2012 Pearson Education, Inc.
DNA Microarrays Allow the
Expression of Thousands of Genes to
Be Monitored Simultaneously
• Northern blotting is used to determine whether a
particular gene is active in a particular cell type or
tissue
• An RNA sample is run on a gel to separate the
molecules by size; the gel is blotted onto a special
paper, which is then exposed to a labeled DNA
probe for the sequence of interest
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Microarrays
• A DNA micorarray is used to monitor expression of
hundreds or thousands of genes simultaneously
• A microarray is a thin chip spotted with thousands of
DNA fragments corresponding to genes of interest
• RNAs are isolated from a cell population and copied
into single-stranded cDNA molecules
© 2012 Pearson Education, Inc.
Microarrays (continued)
• The cDNAs are labeled with a fluorescent dye
• The microarrays are exposed to the labeled DNAs,
which will bind their complementary DNA
• This approach can be used to compare the patterns
of gene expression in different tissues; a green dye
labels cDNAs from one cell type and a red dye the
cDNAs from the other cell type
© 2012 Pearson Education, Inc.
Microarrays (continued)
• The mixture of cDNAs is placed on the microarray,
and binds to genes expressed in each cell type
• Green spots indicate genes expressed only by one
cell type; red spots indicate genes expressed only in
the other cell type
• Genes that are expressed with both types will be
yellow (the color resulting from a mixture of the red
and green labels)
© 2012 Pearson Education, Inc.
Figure 23-19
© 2012 Pearson Education, Inc.
Proximal Control Elements Lie Close
to the Promoter
• The specificity of transcription is determined by
proteins called transcription factors
• General transcription factors are essential for
transcription of all the genes transcribed by a given
type of RNA polymerase
• General transcription factors assemble with RNA
polymerase at the core promoter, very close to the
transcription startpoint
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General transcription factors and
basal transcription
• General transcription factors usually initiate
transcription at low, basal rates
• Most protein coding genes have short DNA
sequences farther upstream to which additional
transcription factors bind, improving the efficiency of
the core promoter
• Proximal control elements are sequences about
100–200 bases upstream of the core promoter
© 2012 Pearson Education, Inc.
Proximal control elements
• The number, exact location and identities of proximal
control elements varies with each gene, but three
types are common
–The CAAT box
–The GC box
–The octamer
• Transcription factors that bind one of these are called
regulatory transcription factors
© 2012 Pearson Education, Inc.
Figure 23-20
© 2012 Pearson Education, Inc.
Enhancers and Silencers Are Located
at Variable Distances from the
Promoter
• Distal control elements, located upstream or
downstream from the genes they regulate, often lie
far away from the promoter
• Enhancers stimulate gene transcription, and
silencers inhibit transcription
• The position of these elements relative to the
promoter can vary greatly
© 2012 Pearson Education, Inc.
Enhancers
• A typical enhancer contains several different
control elements within it, each a short DNA
sequence that is a binding site for a different
regulatory factor
• Some of these sequences may be identical to
proximal control elements
• The regulatory factors that bind enhancers are
called activators
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Studying enhancer function
• To investigate the properties of enhancers,
researchers use recombinant DNA techniques to
alter enhancer location and orientation relative to
the regulated gene
• Enhancers can function at variable distances from
the startpoint if the promoter is present
• When either the promoter or enhancer is missing,
transcription ceases
© 2012 Pearson Education, Inc.
Figure 23-21
© 2012 Pearson Education, Inc.
Silencers and repressors
• Silencers share many features of enhancers, but
inhibit transcription rather than activating it
• Regulatory transcription factors that bind to
silencers are called repressors
• Proteins produced by the SIR gene are examples of
repressors
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Insulators
• Silencers and enhancers can function far from the
genes they regulate
• This creates a complication if genes with different
expression patterns reside near each other
• DNA sequences called insulators are sometimes
employed to prevent an enhancer or silencer from
affecting the wrong genes
© 2012 Pearson Education, Inc.
Coactivators Mediate the Interaction
Between Regulatory Transcription
Factors and the RNA Polymerase
Complex
• Coactivators include chromatin remodeling proteins,
and enzymes that modify histones
• A multiprotein complex called Mediator acts as a bridge
that binds to activator proteins associated with
enhancers and to RNA polymerase
• It links enhancers to components involved in
transcription initiation
© 2012 Pearson Education, Inc.
Triggering gene activation
• Activator proteins bind to their DNA control
elements in the enhancer, forming a complex
called the enhanceosome
• One or more of the activator proteins cause the
DNA to form a loop that brings the enhancer close
to the core promoter (1)
© 2012 Pearson Education, Inc.
Figure 23-22, Steps 1, 2
© 2012 Pearson Education, Inc.
Triggering gene activation (continued)
• The activators interact with proteins such as
chromatin remodeling proteins and HAT, that alter
chromatin structure at the promoter (2)
• Finally the activators bind to Mediator, which
facilitates correct positioning of RNA polymerase
and general transcription factors, allowing
transcription to begin (3)
© 2012 Pearson Education, Inc.
Figure 23-22, Step 3
© 2012 Pearson Education, Inc.
Multiple DNA Control Elements and
Transcription Factors Act in
Combination
• The combinatorial model for gene regulation
proposes that a small number of control elements
and transcription factors act in combinations to
establish patterns of gene expression in different
cell types
• Transcription of genes encoding tissue-specific
proteins requires transcription factors or
combinations of them that are unique to individual
cell types
© 2012 Pearson Education, Inc.
Figure 23-23
© 2012 Pearson Education, Inc.
Figure 23-23A
© 2012 Pearson Education, Inc.
Figure 23-23B
© 2012 Pearson Education, Inc.
Several Common Structural Motifs Allow
Regulatory Transcription Factors to Bind
to DNA and Activate Transcription
• Regulatory transcription factors possess the ability
to bind to a specific DNA sequence and the ability to
regulate transcription
• These activities reside in separate protein domains:
the DNA-binding domain, and the transcription
regulation domain (activation domain)
• Domain swap experiments demonstrate the
independence of the two domains
© 2012 Pearson Education, Inc.
Figure 23B-1
© 2012 Pearson Education, Inc.
Categories of DNA-Binding Domains
• Most regulatory transcription factors can be placed
into one of a small number of categories based on
the motif (secondary structure pattern) that makes
up the DNA binding domain
© 2012 Pearson Education, Inc.
Helix-Turn-Helix Motif
• The helix-turn-helix motif is one of the most
common DNA-binding motifs in both eukaryotic and
prokaryotic regulatory transcription factors
• It consists of two -helices separated by a bend in
the polypeptide chain
• One -helix, the recognition helix, binds the DNA,
and the second helix stabilizes the overall
configuration
© 2012 Pearson Education, Inc.
Figure 23-24A
© 2012 Pearson Education, Inc.
Zinc Finger Motif
• The zinc finger motif consists of an  helix and a
two-segment  sheet held in place by the
interaction of cysteine and histidine residues with a
zinc ion
• Zinc fingers protrude from the protein surface and
act as points of contact between the protein and
the DNA
© 2012 Pearson Education, Inc.
Figure 23-24B
© 2012 Pearson Education, Inc.
Leucine Zipper Motif
• The leucine zipper motif is formed by the
interaction between two polypeptide chains, each
with regularly spaced leucine residues
• The leucine interact with one another and interlock,
causing the two helices to wrap around each other
© 2012 Pearson Education, Inc.
Figure 23-24C
© 2012 Pearson Education, Inc.
Helix-Loop-Helix Motif
• Helix-loop-helix motifs contain a short  helix
connected by a loop to another longer  helix
• They contain hydrophobic regions that usually
connect two polypeptides, either similar or different
© 2012 Pearson Education, Inc.
Figure 23-24D
© 2012 Pearson Education, Inc.
DNA Response Elements Coordinate
the Expression of Nonadjacent Genes
• Eukaryotic cells often need to activate a group of
related genes at the same time
• Eukaryotes use DNA control sequences called
response elements to turn transcription on and off
in response to a particular signal
© 2012 Pearson Education, Inc.
Steroid Hormone Receptors Act as
Transcription Factors That Bind to
Hormone Response Elements
• Steroid receptors and the related retinoid receptors
belong to the zinc finger class of transcription
factors
• They typically consist of three domains: one
recognizes and binds to a specific response
element in DNA, a second binds a particular
hormone, and the third activates transcription
© 2012 Pearson Education, Inc.
Hormone response elements
• Steroid receptors bind to DNA sequences called
hormone response elements
• All genes activated by a particular steroid hormone
are associated with the same type of response
element
• E.g. genes activated by estrogen have an estrogen
response element; genes activated by
glucocorticoids are next to glucocorticoid response
elements
© 2012 Pearson Education, Inc.
Figure 23-25A
© 2012 Pearson Education, Inc.
Cortisol function
• The GR cannot enter the nucleus as long as it is
bound to the Hsp proteins
• Binding of cortisol to the GR triggers release of Hsp
and the GR with the bound cortisol enters the
nucleus to bind glucocorticoid response elements
• Binding of one GR to the response element
facilitates binding of a second
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Cortisol function
• The GR dimer activates transcription of the adjacent
gene by recruiting coactivators that promote
transcription
• The glucocorticoid receptor contains an inverted
repeat, which facilitates binding of two GRs
• Cortisol activates all genes with glucocorticoid
response elements simultaneously
© 2012 Pearson Education, Inc.
CREBs and STATs Are Examples of
Transcription Factors Activated by
Phosphorylation
• Another approach for controlling transcription factor
activity is based on protein phosphorylation
• cAMP in eukaryotes functions by stimulating activity
of protein kinase A, which in turn phosphorylates a
variety of proteins
• One target is CREB, which normally binds DNA
sequences called cAMP response elements
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CREB and CBP
• Once phosphorylated, CREB binds a coactivator
called CBP (CREB-binding protein)
• CBP catalyzes histone acetylation, loosening the
packing of nucleosomes, and facilitates assembly
of the transcription machinery at nearby promoters
© 2012 Pearson Education, Inc.
Figure 23-26
© 2012 Pearson Education, Inc.
STATs
• Another phosphorylation target is a family of
transcription factors called STATs
• Among the signaling molecules that activate
STATs are interferons, glycoproteins secreted by
animal cells in response to viral infections
• Binding of interferon to its cell surface receptor
causes cytosolic activation of the Janus kinase
(Jak)
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STATs
• Activated Jak phosphorylates STAT molecules in
the cytoplasm
• Phosphorylation causes STATs to dimerize and
enter the nucleus where they bind target DNA
response elements
• The Jak-STAT signaling pathway shows
considerable specificity
© 2012 Pearson Education, Inc.
Homeotic Genes Code for
Transcription Factors That Regulate
Embryonic Development
• Homeotic genes are an unusual class of genes
that when mutated cause transformation of one
body structure into another
– E.g., the bithorax gene complex, and the
antennapedia gene complex
• These genes each contain a similar 180-bp
sequence near the 3 end, called the homeobox,
which codes for the homeodomain
© 2012 Pearson Education, Inc.
Figure 23-27
© 2012 Pearson Education, Inc.
Figure 23-27A
© 2012 Pearson Education, Inc.
Figure 23-27B
© 2012 Pearson Education, Inc.
Figure 23-27C
© 2012 Pearson Education, Inc.
Figure 23-28A
© 2012 Pearson Education, Inc.
Homeotic genes
• The homeotic genes code for a family of regulatory
transcription factors that activate or inhibit
transcription of developmentally important genes
• Each homeotic transcription factor can influence
expression of dozens or hundreds of genes in the
growing embryo
© 2012 Pearson Education, Inc.
Figure 23-28B
© 2012 Pearson Education, Inc.
Homeotic genes (continued)
• The result of the coordinated gene regulation by
homeotic genes is the establishment of
fundamental body characteristics such as limb
shape and location
• Genes similar to these are found in a variety of
organisms
• The organization is also quite similar from one
organism to the next
© 2012 Pearson Education, Inc.
Homeotic genes (continued)
• In flies and vertebrates the 3 to 5 location of these
genes corresponds to the anterior to posterior
position along the body axis
• The genes control identity of structures along the
anterior-posterior axis
• E.g., mutations in the HoxD13 locus result in
synpolydactyly, fusions and duplications of fingers
© 2012 Pearson Education, Inc.
Figure 23-29
© 2012 Pearson Education, Inc.
Eukaryotic Gene Regulation:
Posttranscriptional Control
• After transcription has taken place, the flow of
genetic information involves a complex series of
posttranscriptional events
• Posttranscriptional regulation is especially useful in
rapidly fine-tuning patterns of gene expression
© 2012 Pearson Education, Inc.
Control of RNA Processing and Nuclear
Export Follows Transcription
• Posttranscriptional control provides many
opportunities for regulation of gene expression
• RNA splicing is an especially important control
site—its regulation allows cells to create a variety of
different mRNAs from the same pre-mRNA
• Alternative splicing permits some splice sites to be
skipped and others activated
© 2012 Pearson Education, Inc.
Alternative splicing
• Regulatory proteins and small nucleolar RNAs
(snoRNAs) bind to splicing enhancer or splicing
silencer sequences in pre-mRNA
• E.g., the mRNA coding for the immunoglobulin M
(IgM), which has two alternative poly(A) sites
• Alternative splicing leads to the formation of mRNAs
that code for either a secreted or membrane-bound
version of the protein
© 2012 Pearson Education, Inc.
Figure 23-30
© 2012 Pearson Education, Inc.
Nuclear export
• Export of nuclei to the cytoplasm can be
controlled—RNAs with defects in capping or
splicing are not readily exported from the nucleus
• Even with normal mRNAs certain molecules are
retained until their export is triggered by a stimulus
© 2012 Pearson Education, Inc.
Translation Rates Can Be Controlled
by Initiation Factors and
Translational Repressors
• Once mRNAs reach the cytoplasm, several
translational control mechanisms regulate their
rate of translation
• Some mechanisms work by altering ribosomes or
protein synthesis factors
• Others work by regulating the activity/stability of the
mRNA itself
© 2012 Pearson Education, Inc.
A well-studied example of translational
control
• Globin polypeptides are the main product of
translation (>90%) in erythrocytes; their synthesis
depends on the availability of heme
• A protein kinase calledheme-controlled inhibitor
(HCI) is inactive in the presence of heme
• In the absence of heme HCI phosphorylates and
inhibits eIF2, a protein required for initiating
translation
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eIF4F is also regulated by phosphorylation
• eIF4F is a multiprotein complex that binds the 5
mRNA cap and is regulated by phosphorylation
• Adenovirus inhibits protein synthesis in infected
cells by blocking phosphorylation of eIF4F that is
normally required for its activation
• Other viruses produce proteases that cleave eIF4F
and inactivate it; viral mRNAs can still be translated
by one of the eIF4F fragments
© 2012 Pearson Education, Inc.
A more specific type of translational
control
• Ferritin is an iron-storage protein whose synthesis is
selectively stimulated in the presence of iron
• The 5 untranslated leader sequence contains a 28
nucleotide segment, the iron response element
(IRE)
• This element forms a hairpin loop required for
stimulation of production of ferritin
© 2012 Pearson Education, Inc.
Iron regulation of ferritin synthesis
• When iron concentration is low, a protein called IREbinding protein binds to the IRE sequence in the
ferritin mRNA, which prevents formation of an
initiation complex
• When more iron is present the protein binds iron and
undergoes a conformational change and releases
the IRE, allowing translation
• The IRE-binding protein is an example of a
translational repressor
© 2012 Pearson Education, Inc.
Figure 23-31A
© 2012 Pearson Education, Inc.
Figure 23-31B
© 2012 Pearson Education, Inc.
Translation Can Also Be Controlled by
Regulation of mRNA Degradation
• Translation rates are subject to control by
alterations in mRNA stability
• Degradation rates of mRNA can be measured by
pulse-chase experiments
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Pulse-chase experiments
• Cells are incubated in the presence of a radioactive
compound that is incorporated into mRNA, then
placed in a nonradioactive medium
• The fate of radioactivity in the mRNA can be
measured
• The mRNA half-life is the time required for 50% of
the initial radioactive RNA to be degraded
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mRNA stability
• Eukaryotic mRNA stability varies widely
• mRNAs with longer poly(A) tails are more
stable than those with shorter tails
• Short-lived mRNAs have AU-rich elements in
the 3 untranslated region; these trigger
removal of the poly(A) tail
© 2012 Pearson Education, Inc.
Role of iron
• The transferrin receptor mediates iron uptake
across the plasma membrane
• When iron levels are low, the IRE-binding protein
binds an IRE in the 3 UTR of the transferrin
receptor mRNA
• They protect it from degradation
• High iron levels cause the IRE-binding protein to
release the IRE, allowing degradation of the mRNA
© 2012 Pearson Education, Inc.
Figure 23-32A
© 2012 Pearson Education, Inc.
Figure 23-32B
© 2012 Pearson Education, Inc.
Two mechanisms for mRNA degradation
• In the 3 to 5 pathway shortening of the poly(A) tail,
degradation of the RNA chain is accomplished by
the cytoplasmic exosome, a complex of several
exonucleases, with the 5 cap degraded last
• The 5 to 3 pathway involves shortening the tail at
the 3 end, followed by removal of the cap and then
degradation of the mRNA by mRNA processing
bodies (P bodies)
© 2012 Pearson Education, Inc.
RNA Interference Utilizes Small RNAs
to Silence the Expression of Genes
Containing Complementary Base
Sequences
• mRNAs can be controlled by a class of small RNA
molecules that inhibit their expression via
complementarity
• RNAi (RNA interference) is based on the ability of
small RNAs to trigger mRNA degradation
© 2012 Pearson Education, Inc.
RNAi
• Double-stranded RNA (from certain types of viruses
or introduced artificially) knocks down the
expression of specific genes
• First, a cytoplasmic ribonuclease called Dicer
cleaves the double-stranded RNA into short
fragments about 21–22 bp long
• The resulting fragments are called siRNAs (small
interfering or silencing RNAs)
© 2012 Pearson Education, Inc.
RNAi
• The siRNAs combine with a group of proteins to
form an inhibitor of gene expression called RISC
(RNA-induced silencing complex), in this case called
the siRISC
• One of the strands is degraded, and the remaining
one binds the siRISC to a target mRNA by
complementary base pairing
• Slicer cleaves the target mRNA
• In some cases siRISC may enter the nucleus and
silence the gene by DNA methylation
© 2012 Pearson Education, Inc.
Figure 23-33
© 2012 Pearson Education, Inc.
MicroRNAs Produced by Normal
Cellular Genes Silence the Translation
of Developmentally Important
Messenger RNAs
• MicroRNAs (miRNAs) are produced by genes found in
almost all eukaryotes
• These bind to and regulate expression of genes that
are separate from the genes that produce the miRNAs
• miRNAs are initially transcribed into longer molecules
called primary microRNAs (pri-mRNAs), which fold into
hairpin loops
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miRNAs
• Looped pri-miRNAs are converted into mature
miRNAs
• A nuclear enzyme called Drosha cleaves the primiRNAs into smaller hairpins called precursor
miRNAs, pre-miRNAs
• The pre-miRNAs are exported to the cytoplasm,
where Dicer cleaves them to form an miRNA
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miRNAs
• The miRNA forms an miRISC, which inhibits
expression of mRNAs containing sequences
complementary to the miRNA
• mRNAs with fully complementary sequences are
degraded by miRISC
• mRNAs with partially complementary sequences
are translationally inhibited
© 2012 Pearson Education, Inc.
Figure 23-34
© 2012 Pearson Education, Inc.
miRNA roles during development
• MiRNAs play important roles in embryonic
development
• E.g. deleting an miRNA called miR-1-2 causes heart
defects leading to embryonic lethality in mice
• At least a dozen of the mRNAs targeted for
degradation by miR-1-2 have been identified
© 2012 Pearson Education, Inc.
Posttranslational Control Involves
Modification of Protein Structure,
Function, and Degradation
• The posttranslational control mechanisms include
–
–
–
–
Structural alterations that influence protein function
Guiding of protein folding
Targeting to specific locations
Interaction with regulatory molecules
© 2012 Pearson Education, Inc.
Rates of protein synthesis and
degradation
• Relative contributions of rates of protein synthesis
and degradation can be summarized by
– Where P is the concentration of protein, ksyn is the
rate of synthesis, and kdeg is the rate of degradation
© 2012 Pearson Education, Inc.
Rates of protein synthesis and
degradation
• Rates of protein degradation are often expressed as
the half-life of a protein
• Enzymes with short half-lives change more
dramatically in response to alterations in rate of
synthesis than proteins with longer half-lives
• Enzymes important in metabolic regulation tend to
have short half-lives
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Ubiquitin Targets Proteins for
Degradation by Proteasomes
• Ubiquitin is a small protein containing 76 amino
acids
• It is joined to target proteins by a process involving
three components
– A ubiquitin-activating enzyme (E1)
– A ubiquitin-conjugating enzyme (E2)
– A substrate recognition protein, or ubiquitin
ligase (E3)
© 2012 Pearson Education, Inc.
Figure 23-35
© 2012 Pearson Education, Inc.
Proteasomes
• Proteasomes are large protein-degrading structures,
and the predominant proteases of the cytosol; they
bind ubiquitin-labeled proteins and remove the
ubiquitin
• The proteins are then fed into the central channel of
the proteasome and their peptide bonds are
hydrolyzed in an ATP-dependent manner
© 2012 Pearson Education, Inc.
Protein degradation
• Internal amino acid sequences called degrons
target particular proteins for degradation
• Certain N-terminal amino acids of proteins are
sometimes targeted for ubiquitylation
• Proteasomes also play a role in general
mechanisms for eliminating defective proteins from
cells
© 2012 Pearson Education, Inc.
SUMOylation
• Proteins can also be modified by the addition of
small ubiquitin-related modifiers (SUMOs)
• SUMOylation alters protein stability, movement
into and out of the nucleus, and regulation of
transcription factor function
© 2012 Pearson Education, Inc.
Other means of degrading proteins
• Lysosomes take up and degrade proteins by an
infolding of the lysosomal membrane
• Small vesicles are created that are internalized
inside the lysosome and broken down there
• This is microautophagy and the result is a slow
continual recycling of amino acids
© 2012 Pearson Education, Inc.
A Summary of Eukaryotic Gene
Regulation
• There are five distinct levels of regulation possible
–
–
–
–
–
Genomic control
Transcription control
RNA processing and localization
Translational control
Posttranslational control
© 2012 Pearson Education, Inc.