Mod 4 regulation of gene expression -notes SH.pdf

This note is created for BSc students of molecular biology, biochemistry, biotechnology microbiology, and other biological science students.

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Regulation of gene expression in Prokaryotes and Eukaryotes
I. Regulation of gene expression in prokaryotes
Learning Objectives
By the end of this section, you will be able to:
 Explain the process of epigenetic gene regulation in prokaryotic cells.
 Explain the process of transcriptional gene regulation in proaryotic cells.
 Explain the process of regulation Lac-operon.
 Explain the process of regulation of Trp-operon


Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two
pure bacterial cultures inoculated from the same starting colony contain the same DNA, with the exception of
changes that arise from spontaneous mutations. If each cell in a multicellular organism has the same DNA,
then how is it that cells in different parts of the organism’s body exhibit different characteristics? Similarly,
how is it that the same bacterial cells within two pure cultures exposed to different environmental conditions
can exhibit different phenotypes? In both cases, each genetically identical cell does not turn on, or express,
the same set of genes. Only a subset of proteins in a cell at a given time is expressed.
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or
enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a
gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms allows for
cellular differentiation, in single-celled organisms like prokaryotes, it primarily ensures that a cell’s resources
are not wasted making proteins that the cell does not need at that time.
Elucidating the mechanisms controlling gene expression is important to the understanding of human health.
Malfunctions in this process in humans lead to the development of cancer and other diseases. Understanding
the interaction between the gene expression of a pathogen and that of its human host is important for the
understanding of a particular infectious disease. Gene regulation involves a complex web of interactions
within a given cell among signals from the cell’s environment, signaling molecules within the cell, and the
cell’s DNA. These interactions lead to the expression of some genes and the suppression of others, depending
on circumstances.
Prokaryotes and eukaryotes share some similarities in their mechanisms to regulate gene expression; however,
gene expression in eukaryotes is more complicated because of the temporal and spatial separation between
the processes of transcription and translation. Thus, although most regulation of gene expression occurs
through transcriptional control in prokaryotes, regulation of gene expression in eukaryotes occurs at the
transcriptional level and post-transcriptionally (after the primary transcript has been made).

1.1 Prokaryotic Gene Regulation
In bacteria and archaea, structural proteins with related functions are usually encoded together within the
genome in a block called an operon and are transcribed together under the control of a
single promoter, resulting in the formation of a polycistronic transcript (Figure 1.1). In this way, regulation
of the transcription of all of the structural genes encoding the enzymes that catalyze the many steps in a single
biochemical pathway can be controlled simultaneously, because they will either all be needed at the same
time, or none will be needed. For example, in E. coli, all of the structural genes that encode enzymes needed
to use lactose as an energy source are encoded next to each other in the lactose (or lac) operon under the
control of a single promoter, the lac promoter. French scientists François Jacob (1920–201) and

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Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons,
through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or
Medicine in 1965.


Figure 1.1 Schematic Representation of an Operon. In prokaryotes, structural genes of related functions
are often organized together on the genome and transcribed together under the control of a single promoter.
The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the
operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory
region, enhancing transcription.
Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

Each operon includes DNA sequences that influence its own transcription; these are located in a region called
the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter,
to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence
the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes.
A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus
by binding to a DNA sequence within the regulatory region called the operator, which is located between the
RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene.
Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely,
an activator is a transcription factor that increases the transcription of a gene in response to an external
stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory
molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or
an activator.
In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose
expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed
and translated continuously to provide the cell with constant intermediate levels of the protein products. Such
genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA
replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other
prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and
inducers.
Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby
preventing the transcription of the structural genes. Such operons are classified as either repressible
operons or inducible operons. Repressible operons, like the tryptophan (trp) operon, typically contain genes
encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan,
continues to be required by the cell, a repressible operon will continue to be expressed. However, when the

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product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue
to make more, the expression of the operon is repressed. Conversely, inducible operons, like
the lac operon of E. coli, often contain genes encoding enzymes in a pathway involved in the metabolism of
a specific substrate like lactose. These enzymes are only required when that substrate is available, thus
expression of the operons is typically induced only in the presence of the substrate.
The trp Operon: A Repressible Operon
E. coli can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each
other in the trp operon (Figure 1.2). When environmental tryptophan is low, the operon is turned on. This
means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if
tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur and
tryptophan is not synthesized.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the
operon is active and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two
tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to
the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase
from transcribing the structural genes, stopping expression of the operon. Thus, the actual product of the
biosynthetic pathway controlled by the operon regulates the expression of the operon.
Figure 1.2
The Trp Operon. The five structural genes needed to synthesize tryptophan in E. coli are located next to each
other in the trp operon. When tryptophan is absent, the repressor protein does not bind to the operator, and the
genes are transcribed. When tryptophan is plentiful, tryptophan binds the repressor protein at the operator
sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes.
Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The Lac Operon: An Inducible Operon
The lac operon is an example of an inducible operon that is also subject to activation in the absence of
glucose. The lac operon encodes three structural genes, lacZ, lacY, and lacA, necessary to acquire and process
the disaccharide lactose from the environment (Fig 1.3A).

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Figure 1.3 Biological Activity of the lac Operon. (A) Schematic representation of the lac operon in E.
coli. The lac operon has three structural genes, lacZ, lacY, and lacA that encode for β-galactosidase, permease,
and galactoside acetyltransferase, respectively. The promoter (p) and operator (o) sequences that control the

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expression of the operon are shown. Upstream of the lac operon is the lac repressor gene, lacI, controlled by
the lacI promoter (p). (B) Shows the lac repressor inhibition of the lac operon gene expression in the absence
of lactose. The lac repressor binds with the operator sequence of the operon and prevents the RNA polymerase
enzyme which is bound to the promoter (p) from initiating transcription. (C) In the presence of lactose, some
of the lactose is converted into allolactose, which binds and inhibits the activity of the lac repressor.
The lac repressor-allolactose complex cannot bind with the operator region of the operon, freeing the RNA
polymerase and causing the initiation of transcription. Expression of the lac operon genes enables the
breakdown and utilization of lactose as a food source within the organism.
Figure modified from Esmaeili, A., et. al. (2015) BMC Bioinformatics 16:311

The lacZ gene encodes the β-galactosidase (β-gal) enzyme responsible for the hydrolysis of lactose into simple
sugars glucose and galactose (Fig. 1.4A). The β-gal enzyme can also mediate the breakdown of the alternate
substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) (Fig. 1.4B). The breakdown product, 5-
bromo-4-chloro-3-hydroxyindole – 1, spontaneously dimerizes to form the intensely blue blue product, 5,5′-
dibromo-4,4′-dichloro-indigo – 2. Thus, Xgal has been a valuable research tool, not only in the study of the
enzymatic activity of β-gal, but also in the development of the commonly used blue-white DNA cloning
system that utilizes the β-gal enzyme as a marker in molecular cloning experiments.
The lac operon contains two more genes, in addition to lacZ (Fig. 1.3A). The lacY gene encodes a permease
that increases the uptake of lactose into the cell and lacA encodes a galactoside acetyltransferase (GAT)
enzyme. The exact function of GAT during lactose metabolism has not been conclusively elucidated but
acetylation is thought to play a role in the transport of the modified sugars.

Figure 1.4 Reactions Controlled by the Expression of the Lac Operon. (A) Expression of the β-
galactosidase enzyme enables the breakdown of lactose into the simple sugars, glucose, and galactose for E.
coli to use as a food resource. (B) The β-galactosidase enzyme also mediates the breakdown of the non-native
substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal). Breakdown product (1) 5-bromo-4-
chloro-3-hydroxyindole quickly dimerizes into the intensely blue product (2) 5,5′-dibromo-4,4′-dichloro-
indigo making it a useful tool for molecular biology. (C) β-D-1-thiogalactopyranoside (IPTG) can serve as a
non-native inducer of the lac operon. It mimics the structure of lactose and binds with the Lac Repressor.
Figure modified from: Andreas Piehler, Yikrazuul, and NUROtiker

For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be
energetically wasteful to create the enzymes to process lactose if lactose was not available.
In the absence of lactose, the lacI gene is constituitively expressed, expressing the lac repressor protein (Fig.
1.3 B). The lac repressor binds with an operator region of the lac operon and physically prevents RNA

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polymerase from transcribing the structural genes (Fig. 1.3 B). However, when lactose is present, the lactose
inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to
the repressor and changing its shape so that it is no longer able to bind to the operator DNA (Fig. 1.3 C).
Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator
region and begin transcription of the lac structural genes. In addition to lactose, laboratory experiments have
revealed that the non-natural compound Isopropyl β-D-1-thiogalactopyranoside (IPTG) can also bind with
the lac repressor and cause the expression of lac operon (Figure 1.4 C). Similar to Xgal, this compound has
also been used as a research tool for molecular cloning.
The Lac Operon: Activation by Catabolite Activator Protein
Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose
is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are
only used when glucose has been depleted. Additionally, bacteria have mechanisms to ensure that the genes
encoding enzymes for using alternative substrates are expressed only when the alternative substrate is
available. In the 1940s, Jacques Monod was the first to demonstrate the preference for certain substrates over
others through his studies of E. coli’s growth when cultured in the presence of two different substrates
simultaneously. Such studies generated diauxic growth curves, like the one shown in Figure 1.5. Although the
preferred substrate glucose is used first, E. coli grows quickly and the enzymes for lactose metabolism are
absent. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes
needed for the metabolism of the second substrate, lactose. Notice how the growth rate in lactose is slower, as
indicated by the lower steepness of the growth curve.

Figure 1.5. Utilization of Glucose in E. Coli. When grown in the presence of two substrates, E. coli uses the
preferred substrate (in this case glucose) until it is depleted. Then, enzymes needed for the metabolism of the
second substrate are expressed and growth resumes, although at a slower rate.
Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

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The ability to switch from glucose use to another substrate like lactose is a consequence of the activity of an
enzyme called Enzyme IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism and
EIIA becomes phosphorylated. Phosphorylated EIIA activates adenylyl cyclase, an enzyme that converts some
of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of AMP and important signaling molecule
involved in glucose and energy metabolism in E. coli (Fig. 1.6). As a result, cAMP levels begin to rise in the
cell. This is an indicator to the cell, that overall energy levels are low and that ATP is being depleted.
Figure 1.6.
Conversion of ATP to cAMP. When ATP levels decrease due to depletion of glucose, some remaining ATP
is converted to cAMP by adenylyl cyclase. Thus, increased cAMP levels signal glucose depletion.
Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The lac operon also plays a role in this switch from using glucose to using lactose. When glucose is scarce,
the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein
(CAP), also known as cAMP receptor protein (CRP). The complex binds to the promoter region of
the lac operon (Figure 1.7). In the regulatory regions of these operons, a CAP binding site is located upstream
of the RNA polymerase binding site in the promoter. The binding of the CAP-cAMP complex to this site
increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the
structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present
(removing the lac repressor protein) and glucose levels must be depleted (allowing binding of an activating
protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes for the
metabolism of alternative substrates. Because of low cAMP levels under these conditions, there is an
insufficient amount of the CAP-cAMP complex to activate the transcription of these operons.

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Figure 1.7 Effect of CAP on the Lac Operon. (a) In the presence of cAMP, CAP binds to the promoters of
operons, like the lac operon, that encode genes for enzymes for the use of alternate substrates. (b) For
the lac operon to be expressed, there must be activation by cAMP-CAP as well as removal of the lac repressor
from the operator.
Figure from: Parker, N., et. al. (2019) Microbiology. Openstax
Prokaryotic Attenuation and Riboswitches
Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also
mechanisms to control both the completion of transcription, as well as translation, concurrently. Since their
discovery, these mechanisms have been shown to control the completion of transcription and translation of
many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation
directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes.
One such regulatory system is attenuation, whereby secondary stem-loop structures formed within the 5’
end of an mRNA being transcribed determine if transcription to complete the synthesis of this mRNA will
occur and if this mRNA will be used for translation. Beyond the transcriptional repression mechanism already
discussed, attenuation also controls expression of the trp operon in E. coli (Fig. 1.11). The trp operon
regulatory region contains a leader sequence called trpL between the operator and the first structural gene,
which has four stretches of RNA that can base pair with each other in different combinations. When a
terminator stem-loop forms, transcription terminates, releasing RNA polymerase from the mRNA. However,
when an antiterminator stem-loop forms, this prevents the formation of the terminator stem-loop, so RNA
polymerase can transcribe the structural genes.

Figure 1.11. Attenuation of Transcription and Translation. When tryptophan is plentiful, translation of
the short leader peptide encoded by trpL proceeds, the terminator loop between regions 3 and 4 forms, and
transcription terminates. When tryptophan levels are depleted, translation of the short leader peptide stalls at
region 1, allowing regions 2 and 3 to form an antiterminator loop, and RNA polymerase can transcribe the
structural genes of the trp operon.

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Figure from: Parker, N., et. al. (2019) Microbiology. Openstax
II.Eukaryotic Gene Regulation
Learning Objectives
By the end of this section, you will be able to:
 Explain the process of epigenetic gene regulation in eukaryotic cells.
 Explain the process of transcriptional gene regulation in eukaryotic cells.
 Explain the process of post-transcriptional gene regulation in eukaryotic cells.
 Explain the process of translational gene regulation in eukaryotic cells.
 Explain the process of post-transcriptional gene regulation in eukaryotic cells.
In eukaryotes, control of gene expression is more complex and can happen at many different
levels. Eukaryotic genes are not organized into operons, so each gene must be regulated
independently. In addition, eukaryotic cells have many more genes than prokaryotic cells.
Regulation of gene expression can happen at any of the stages as DNA is transcribed into
mRNA and mRNA is translated into protein. For convenience, regulation is divided into five
levels: epigenetic, transcriptional, post-transcriptional, translational, and post-translational
(Figure 1 below).


Figure: 1Regulation of gene expression in eukaryotes can occur at five different levels. Here,
the Central Dogma is diagrammed with arrows showing where each type of eukaryotic
regulation of gene expression interrupts it.

Compared to prokaryotes, many steps in eukaryotes lie between the transcription of an
mRNA and the accumulation of a polypeptide end product. Eleven of these steps are shown
in the pathway from gene to protein below.

Theoretically, cells could turn on, turn off, speed up or slow down any of the steps in this
pathway, changing the steady state concentration of a polypeptide in the cells. While
regulation of any of these steps is possible, the expression of a single gene is typically
controlled at only one or a few steps. A common form of gene regulation is at the level of
transcription initiation, similar to transcriptional control in bacteria, in principle if not in
detail. (Refer to fig 1.1below)

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Fig 1.1
Epigenetic Control of Gene Expression
The first level of control of gene expression is epigenetic (“around genetics”) regulation.
Epigenetics is a relatively new, but growing, field of biology.
Epigenetic control involves changes to genes that do not alter the nucleotide sequence of the
DNA and are not permanent. Instead, these changes alter the chromosomal structure so that
genes can be turned on or off. This level of control occurs through heritable chemical
modifications of the DNA and/or chromosomal proteins.
One example of chemical modifications of DNA is the addition of methyl groups to the DNA,
in a process called methylation, In general, methylation suppresses transcription.
Interestingly, methylation patterns can be passed on as cells divide. Thus, parents may be
able to pass on the tendency of a gene to be expressed in their offspring. Other heritable
chemical modifications of DNA may also occur.

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Modification of Histone Proteins is an Example of Epigenetic Control
The best-studied example of epigenetic regulation is a modification of histone proteins.
Histones are chromosomal proteins that tightly wind DNA so that it fits into the nucleus of a
cell. The human genome, for example, consists of over three billion nucleotide pairs. An
average chromosome contains 10 million nucleotide pairs, and each body cell contains 46
chromosomes. If stretched out linearly, an average human chromosome would be over four
centimeters long. In order to fit all of this DNA into the nucleus of a microscopic cell, the DNA
must be tightly wound around proteins. It is also organized so that specific segments can be
accessed as needed by a specific cell type (Figure 2).


Figure 2 In each chromosome, DNA is wound around histone proteins to pack it into the
nucleus of a cell. (Credit: modification of work by NIH.)
The first level of organization, or packing, is the winding of DNA strands around histone
proteins. Histones package and order DNA into structural units called nucleosome
complexes, which can control the access of proteins to the DNA regions (Figure 3). Under the
electron microscope, this winding of DNA around histone proteins to form nucleosomes

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looks like small beads on a string (Figure 4). These beads (histone proteins) can move along
the string (DNA) and change the structure of the molecule.
Figure 4, DNA is wrapped around histones to create nucleosomes (a), which control the
access of proteins to DNA. When viewed through an electron microscope (b), the
nucleosomes look like beads on a string. (Credit “micrograph”: modification of work by Chris
Woodcock.)
If a gene is to be transcribed, the nucleosomes surrounding that region of DNA can slide down
the DNA to open that specific chromosomal region and allow access for RNA polymerase and
other proteins, called transcription factors, to bind to the promoter region and initiate
transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA
have different modifications that signal a closed chromosomal configuration. In this closed
configuration, the RNA polymerase and transcription factors do not have access to the DNA
and transcription cannot occur (Figure 4).
How the histone proteins move is dependent on signals found on the histone proteins. These
signals are “tags” – in the form of phosphate, methyl, or acetyl groups – that open or close a
chromosomal region (Figure 4). These tags are not permanent but may be added or removed
as needed. Since DNA is negatively charged, changes in the charge of the histone will change
how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a
large positive charge; by adding chemical modifications like acetyl groups, the charge
becomes less positive.

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Figure 4 Nucleosomes can slide along DNA. (A) When nucleosomes are spaced closely
together, transcription factors cannot bind and gene expression is turned off. (B) When
nucleosomes are spaced far apart, transcription factors can bind, allowing gene expression
to occur.
Regulating Eukaryotic Genes Means Contending with Chromatin
Consider again the illustration of the different levels of chromatin structure (below).

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Transcription factors bind specific DNA sequences by detecting them through the grooves
(mainly the major groove) in the double helix. The drawing above reminds us however, that
unlike the nearly naked DNA of bacteria, eukaryotic (nuclear) DNA is coated with proteins
that, in aggregate are by mass, greater than the mass of DNA that they cover. The protein-
DNA complex of the genome is of course, chromatin.
Again, as a reminder, DNA coated with histone proteins forms the 9 nm diameter beads-on-
a-string structure in which the beads are the nucleosomes. The association of specific non-
histone proteins causes the nucleosomes to fold over on themselves to form the 30 nm
solenoid. As we saw earlier, it is possible to selectively extract chromatin. Take a second look
at the results of typical extractions of chromatin from isolated nuclei below.
Further accretion of non-histone proteins leads to more folding and the formation
of euchromatin and heterochromatin characteristic of non-dividing cells. In dividing cells,
the chromatin further condenses to form the chromosomes that separate during
either mitosis or meiosis.

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Recall that biochemical analysis of the 10 nm filament extract revealed that the DNA wraps
around histone protein octamers, the nucleosomes or beads in this beads-on-a string
structure. Histone proteins are highly conserved in eukaryotic evolution (they are not found
in prokaryotes). They are also very basic (many lysine and arginine residues) and therefore
very positively charged. This explains why they are able to arrange themselves uniformly
along DNA, binding to the negatively charged phosphodiester backbone of DNA in the double
helix.
Since the DNA in euchromatin is less tightly packed than it is in heterochromatin, perhaps
active genes are to be found in euchromatin and not in heterochromatin. Experiments in
which total nuclear chromatin extracts were isolated and treated with the enzyme
deoxyribonuclease (DNAse) revealed that the DNA in active genes was degraded more rapidly
than non-transcribed DNA. More detail on these experiments can be found in the two links
below.
The results of such experiments are consistent with the suggestion that active genes are
more accessible to DNAse because they are in less coiled, or less condensed chromatin. DNA
in more condensed chromatin is surrounded by more proteins, and thus is less accessible to,
and protected from DNAse attack. When packed up in chromosomes during mitosis or
meiosis, all genes are largely inactive.
Regulating gene transcription must occur in non-dividing cells or during the interphase of
cells, where changing the shape of chromatin (chromatin remodeling) in order to silence
some and activate other genes is possible. Changing chromatin conformation involves the
chemical modification of chromatin proteins and DNA. For example, chromatin can be
modified by histone acetylation, de-acetylation, methylation and phosphorylation, reactions
catalyzed by histone acetyltransferases (HAT enzymes), de-acetylases, methyl transferases
and kinases, respectively. For example, acetylation of lysines near the amino end of histones
H2B and H4 tends to unwind nucleosomes and open the underlying DNA for transcription.
De-acetylation then, promotes condensation of the chromatin in the affected regions of DNA.
Likewise, methylation of lysines or arginines (the basic amino acids that characterize
histones!) of H3 and H4 can open DNA for transcription, while demethylation has the
opposite effect. In one case, di-methylation of a lysine in H3 can suppress transcription. These
chemical modifications affect recruitment of other proteins that alter chromatin
conformation and ultimately activate or block transcription.
This reversible and its effect on chromatin are illustrated below.

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Nucleosomes themselves can be moved, slid and otherwise repositioned by complexes that
hydrolyze ATP for energy to accomplish the physical shifts. Some cancers are associated with
mutations in genes for proteins involved in chromatin remodeling. This is no doubt, because
failures of normal remodeling could adversely affect normal cell cycling and normal
replication. In fact, a single, specific pattern of methylation may mark DNA in multiple cancer
types (check out Five Cancers with the Same Genomic Signature - Implications).
Transcriptional Control of Gene Expression
Transcriptional regulation is the control of whether or not an mRNA is transcribed from a
gene in a particular cell. Like prokaryotic cells, the transcription of genes in eukaryotes
requires an RNA polymerase to bind to a promoter to initiate transcription. In eukaryotes,
RNA polymerase requires other proteins, or transcription factors, to facilitate transcription
initiation. Transcription factors are proteins that bind to the promoter sequence and other
regulatory sequences to control the transcription of the target gene. RNA polymerase by
itself cannot initiate transcription in eukaryotic cells. Transcription factors must bind to the
promoter region first and recruit RNA polymerase to the site for transcription to begin.
The Promoter and Transcription Factors
In eukaryotic genes, the promoter region is immediately upstream of the coding sequence.
This region can range from a few to hundreds of nucleotides long. The length of the promoter
is gene-specific and can differ dramatically between genes. The longer the promoter, the
more available space for proteins to bind. Consequently, the level of control of gene
expression can differ quite dramatically between genes. The purpose of the promoter is to
bind transcription factors that control the initiation of transcription (Figure 17.10, top).
Within the promoter region, just upstream of the transcriptional start site, resides the TATA
box. This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA
repeats). Transcription factors bind to the TATA box, assembling an initiation complex. Once
this complex is assembled, RNA polymerase binds to its upstream sequence and becomes
phosphorylated. This releases part of the protein from the DNA, activates the transcription

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initiation complex, and places RNA polymerase in the correct orientation to begin
transcription (Figure 5, top).


Figure 5 Top. Each gene has a promoter upstream of the coding sequence. The promoter
binds to transcription factors and helps RNA polymerase to bind and start transcription.
Bottom. Many genes also have upstream enhancers. Enhancers bind activators, bend around,
and help RNA polymerase start transcription.
Enhancers and Repressors
In some eukaryotic genes, there are regions that help increase transcription. These regions,
called enhancers, are not necessarily close to the genes; they can be located thousands of
nucleotides away. They can be found upstream, within the coding region, or downstream
of a gene. Enhancers are binding sites for activators. When an enhancer is far away from a
gene, the DNA folds such that the enhancer is brought into proximity with the promoter,
allowing interaction between the activators and the transcription initiation complex
(Figure 5,)
Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription.
Transcriptional repressors can bind to promoter or enhancer regions and block transcription.
Both activators and repressors respond to external stimuli to determine which genes need
to be expressed.

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Post-transcriptional Control of Gene Expression
Post-transcriptional regulation occurs after the mRNA is transcribed but before translation
begins. This regulation can occur at the level of mRNA processing, transport from the nucleus
to the cytoplasm, or binding to ribosomes.
Alternative RNA splicing
In eukaryotic cells the RNA primary transcript often contains introns, which are removed
prior to translation.
Alternative RNA splicing is a mechanism that allows different combinations of introns, and
sometimes exons, to be removed from the primary transcript (Figure 6). This allows different
protein products to be produced from one gene. Alternative splicing can act as a mechanism
of gene regulation. Differential splicing is used to produce different protein products in
different cells or at different times within the same cell. Alternative splicing is now
understood to be a common mechanism of gene regulation in eukaryotes; up to 70 percent
of genes in humans are expressed as multiple proteins through alternative splicing.

Figure 6 Before a RNA can be translated, introns must be removed by splicing. Pre-mRNA can
be alternatively spliced to create different proteins.
Evolution of Alternative Splicing
How could alternative splicing evolve? Introns have a beginning and ending recognition
sequence; it is easy to imagine the failure of the splicing mechanism to identify the end of an
intron and instead find the end of the next intron, thus removing two introns and the
intervening exon. In fact, there are mechanisms in place to prevent such intron skipping, but
mutations are likely to lead to their failure. Such “mistakes” would more than likely produce
a non-functional protein. Indeed, the cause of many genetic diseases is alternative splicing
rather than mutations in a sequence. However, alternative splicing would create a protein
variant without the loss of the original protein, opening up possibilities for adaptation of the
new variant to new functions. Gene duplication has played an important role in the evolution
of new functions in a similar way by providing genes that may evolve without eliminating the
original, functional protein.

19. SARDAR HUSSAIN 19

Control of RNA Stability
Another type of post-transcriptional control involves the stability of the mRNA in the
cytoplasm. The longer an mRNA exists in the cytoplasm, the more time it has to be translated,
and the more protein is made. Many factors contribute to mRNA stability, including the
length of its poly-A tail.

Figure 7: The
protein-coding region of mRNA is flanked by 5′ and 3′ untranslated regions (UTRs). RNA-
binding proteins at the 5′ or 3′ UTR influence the stability of the RNA molecule.
Proteins, called RNA-binding proteins (RBPs) can bind to the regions of the RNA just upstream
or downstream of the protein-coding region. These regions in the RNA that are not translated
into protein are called the untranslated regions, or UTRs. The region just before the protein-
coding region is called the 5′ UTR, whereas the region after the coding region is called the 3′
UTR (Figure 7). The binding of RBPs to these regions can increase or decrease the stability of
an RNA molecule, depending on the specific RBP that binds.
microRNAs, or miRNAs, can also bind to the RNA molecule. miRNAs are short (21–24
nucleotides) RNA molecules that are made in the nucleus as longer pre-miRNAs and then
chopped into mature miRNAs by a protein called dicer. miRNAs bind to mRNA along with a
ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The RISC-
miRNA complex rapidly degrades the target mRNA.
Translational Control of Gene Expression
After an mRNA has been transported to the cytoplasm, it is translated into proteins. Control
of this process is largely dependent on the mRNA molecule. As previously discussed, the
stability of the mRNA will have a large impact on its translation into a protein. Translation
can also be regulated at the level of binding of the mRNA to the ribosome. Once the mRNA
bound to the ribosome, the speed and level of translation can still be controlled. An example
of translational control occurs in proteins that are destined to end up in an organelle called
the endoplasmic reticulum (ER). The first few amino acids of these proteins are a tag called a
signal sequence. As soon as these amino acids are translated, a signal recognition particle
(SRP) binds to the signal sequence and stops translation while the mRNA-ribosome complex
is shuttled to the ER. Once they arrive, the SRP is removed and translation resumes.

20. SARDAR HUSSAIN 20

Post-translational Control of Gene Expression
The final level of control of gene expression in eukaryotes is post-translational regulation.
This type of control involves modifying the protein after it is made, in such as way as to affect
its activity. One example of post-translational regulation is enzyme inhibition. When an
enzyme is no longer needed, it is inhibited by a competitive or allosteric inhibitor, which
prevents it from binding to its substrate. The inhibition is reversible, so that the enzyme can
be reactivated later. This is more efficient than degrading the enzyme when it is not needed
and then making more when it is needed again.
The activity and/or stability of proteins can also be regulated by adding functional groups,
such as methyl, phosphate, or acetyl groups. Sometimes these modifications can regulate
where a protein is found in the cell—for example, in the nucleus, the cytoplasm, or attached
to the plasma membrane.
The addition of a ubiquitin group to a protein mark that protein for degradation. Ubiquitin
acts like a flag indicating that the protein’s lifespan is complete. Tagged proteins are moved
to a proteasome, an organelle that degrades proteins (Figure 8). One way to control gene
expression, therefore, is to alter the longevity of the protein.

Figure 8 Proteins with ubiquitin tags are marked for degradation within the proteasome.
Ferritin RNA regulation
Iron toxicity in mammals-key points.
• Another way to regulate translation involves RNA-binding proteins that directly affect translational
initiation
• Iron is a vital cofactor for many cellular enzymes
• However, it is toxic at high levels
• To prevent toxicity, mammalian cells synthesize a protein called ferritin, which forms a hollow,
spherical complex that can store excess iron

21. SARDAR HUSSAIN 21

• mRNA that encodes ferritin is controlled by an RNA-binding protein known as the iron regulatory
protein (IRP)

Gene expression is primarily regulated at the pre-transcriptional level, but there are a
number of mechanisms for the regulation of translation as well. One well-studied animal
system is the iron-sensitive RNA-binding protein, which regulates the expression of genes
involved in regulating intracellular levels of iron ions. Two of these genes, ferritin, which
safely sequesters iron ions inside cells, and transferrin, which transports iron from the blood
into the cell, both utilize this translational regulation system in a feedback loop to respond
to intracellular iron concentration, but they react in opposite ways. The key interaction is
between the iron response elements (IRE), which are sequences of mRNA that form short
stem-loop structures, and IRE-BP, the protein that recognizes and binds to the IREs. In the
case of the ferritin gene, the IRE sequences are situated upstream of the start codon. When
there is high iron, the IRE-BP is inactive, and the stem-loop structures are melted and overrun
by the ribosome, allowing translation of ferritin, which is an iron-binding protein. As the iron
concentration drops, the IRE-BP is activated and binds around the IRE stem-loop structures,
stabilizing them and preventing the ribosome from proceeding. This prevents the production
of ferritin when there is little iron to bind.

22. SARDAR HUSSAIN 22

Transferrin also uses iron response elements and IRE-binding proteins but in a very different
mechanism. The IRE sequences of the transferrin gene are located downstream of
the stop codon and play no direct role in allowing or preventing translation.
However, when there is low intracellular iron and there is a need for more transferrin to bring
iron into the cell, the IRE-BP is activated as in the previous case, and it binds to the IREs to
stabilize the stem-loop structures. In this case; however, it prevents the 3’ poly-A tail
degradation that would normally occur over time. Once the poly-A tail is degraded, the rest
of the mRNA is destroyed soon thereafter. As mentioned in the transcription chapter, the
longer poly-A tails are associated with greater persistence in the cytoplasm, allowing more
translation before they are destroyed. The IRE-BP system in this case externally prolongs the
lifetime of the mRNA when that gene product is needed in higher amounts.
Since mRNA is a single-stranded nucleic acid and thus able to bind complementary sequence,
it is not too surprising to find that one of the ways that a cell can regulate translation is using
another piece of RNA. Micro RNAs (miRNAs) were discovered as very short (~20 nucleotides)
non-protein-coding genes in the nematode, C. elegans. Since their initial discovery (Lee et
al, Cell 75: 843-54, 1993), hundreds have been found in various eukaryotes, including
humans. The expression pattern of the miRNA genes is highly specific to tissue and
developmental stage. Many are predicted to form stem-loop structures, and appear to
hybridize to 3’-untranslated sequences of mRNA thus blocking initiation of translation on
those mRNA molecules. They may also work through a mechanism similar to the siRNA
discussed below, but there is clear evidence that mRNA levels are not necessarily altered by
miRNA-directed translational control.
Micro RNAs

23. SARDAR HUSSAIN 23

MicroRNAs are currently under investigation for their roles as either oncogenes or tumor
suppressors (reviewed in Garzon et al, Ann. Rev. Med. 60: 167-79, 2009). Approximately half
of known human miRNAs are located at fragile sites, breakpoints, and other regions
associated with cancers (Calin et al, Proc. Nat. Acad. Sci. (USA) 101: 2999-3004, 2004). For
example, miR-21 is not only upregulated in a number of tumors, its overexpression blocks
apoptosis - a necessary step to allow abnormal cells to continue to live and divide rather than
die out. Conversely, miR-15a is significantly depressed in some tumor cells, and
overexpression can slow or stop the cell cycle, even inducing apoptosis.
Another mechanism for translational control that uses small RNA molecules is RNA
interference (RNAi). This was first discovered as an experimentally induced repression of
translation when short double-stranded RNA molecules, a few hundred nucleotides in length
and containing the same sequence as a target mRNA, were introduced into cells. The effect
was dramatic: most of the mRNA with the target sequence was quickly destroyed. The
current mechanistic model of RNAi repression is that first, the double-stranded molecules are
cleaved by an endonuclease called Dicer, which cleaves with over-hanging single-stranded 3’
ends. This allows the short fragments (siRNA, ~20nt long) to form a complex with several
proteins (RISC, RNA-induced silencing complex). The RISC splits the double-stranded
fragments into single strands, one of which is an exact complement to the mRNA. Because of
the complementarity, this is a stable interaction, and the double-stranded region appears to
signal an endonuclease to destroy the mRNA/siRNA hybrid.
The final method of controlling levels of gene expression is controlled after the fact, i.e., by
targeted destruction of the gene product protein. While some proteins keep working until
they fall apart, others are only meant for short-term use (e.g., to signal a short phase in the
cell cycle) and need to be removed for the cell to function properly. Removal, in this sense,
would be a euphemism for chopped up and recycled. The ubiquitin-proteasome system is a
tag-and-destroy mechanism in which proteins that have outlived their usefulness are
polyubiquitinated. Ubiquitin is a small (76 amino acids, ~5.6 kDa), highly conserved (96%
between human and yeast sequences) eukaryotic protein (Figure 10 ) that can be attached
to other proteins through the action of three sequential enzymatic steps, each catalyzed by
a different enzyme.
E1 activates the ubiquitin by combining it with ATP to make ubiquitin-adenylate and then
transfers the ubiquitin to itself via a cysteine thioester bond. Through a trans(thio)
esterification reaction, the ubiquitin is then transferred to a cysteine in the E2 enzyme, also
known as the ubiquitin-conjugating enzyme. Finally, E3, or ubiquitin ligase, interacts with
both E2-ubiquitin and the protein designated for destruction, transferring the ubiquitin to
the target protein. After several rounds, the polyubiquitinated protein is sent to the
proteasome for destruction.
Mutations in E3 genes can cause a variety of human medical disorders such as the
neurodevelopmental disorders Angelman syndrome, Hippel-Lindau syndrome, or the general

24. SARDAR HUSSAIN 24

growth disorder known as 3-M syndrome. Mechanisms linking malfunction in ubiquitination
pathways and symptoms of these disorders are not currently known.

Figure.11 Polyubiquitination of a targeted protein (blue) requires three ubiquitinating
enzymes, E1, E2, and E3. Once tagged, the protein is positioned in the proteasome by binding
of the polyubiquitin tail to the outer surface of the proteasome. The proteasome then cleaves
the protein into small polypeptides.
Proteasomes are very large protein complexes arranged as a four-layered barrel (the 20S
subunit) capped by a regulatory subunit (19S) on each end. The two outer rings are each
composed of 7 α subunits that function as entry gates to the central rings, each of which is
composed of 7 β subunits, and which contain along the interior surface, 6 proteolytic sites.
The 19S regulatory units control the opening and closing of the gates into the 20S catalytic
barrel. The entire proteasome is sometimes referred to as a 26S particle.
A polyubiquitinated protein is first bound to the 19S regulatory unit in an ATP-dependent
reaction (the 19S contains ATPase activity). 19S unit opens the gates of the 20S unit, possibly
involving ATP hydrolysis, and guides the protein into the central proteolytic chamber. The
protease activity of proteasomes is unique in that it is a threonine protease, and it cuts most
proteins into regular 8-9 residue polypeptides, although this can vary.
As we will see in the cell cycle, proteasomes are a crucial component to precise regulation of
protein functions.
References
 Chapter 17. Regulation of Gene Expression – Introduction to Molecular and Cell Biology (pressbooks.pub)
 10.8: Regulation of Translation - Biology LibreTexts
 Chapter 13: Transcriptional Control and Epigenetics - Chemistry (wou.edu)