Regulation of Gene

Expression 38
Learning Objectives

At the end of this chapter, the student will be able to:

• Describe basic concepts of prokaryotic gene expression like lac operon and molecular switch
• Describe the special features of eukaryotic gene expression
• Explain DNA motifs
• Describe how knowledge of gene expression can be used for medical care

I. OVERVIEW gene expression is best understood in prokaryotes, but

many themes are repeated in eukaryotes. Figure 38.1
Gene expression refers to the multistep process that shows some of the sites where gene expression can be
ultimately results in the production of a functional controlled.
gene product, either ribonucleic acid (RNA) or protein. We can understand some key concepts of gene expres-
The first step in gene expression, the use of deoxyribonu- sion by first understanding the response of gene to a
cleic acid (DNA) for the synthesis of RNA (transcription), signal (type A, B, and C responses), lac operon system
is the primary site of regulation in both prokaryotes and in prokaryotes (an example of de-repression), tryptophan
eukaryotes. In eukaryotes, however, gene expression also operon in prokaryotes (example of control by attenuation)
involves extensive posttranscriptional and posttransla- and the prokaryotic molecular switch (genes regulating
tional processes as well as actions that influence access each other’s expression).
to particular regions of the DNA. Each of these steps can Type A response to a gene signal implies that there will
be regulated to provide additional control over the kinds be increased expression of a gene only till the signal is
and amounts of functional products that are produced. present. Once the signal is removed, expression of the
All somatic cells have same genes with the exception of gene will come back to basal levels. Prokaryotic response
amplified and rearranged genes. Organisms adapt to envi- to nutrients and eukaryotic response to hormones are
ronmental (cellular and external) changes by altering gene examples of type A response. In type B response,
expression. Cells do not express all their genes. Also, increased amount of gene expression is transient even in
not all genes are tightly regulated. For example, genes the continued presence of regulatory signal. Some devel-
described as constitutive encode products required for opmental gene expressions follow type B responses.
basic cellular functions and so are expressed at essentially Type C response, like response to Cro protein, is the
a constant level. They are also known as “housekeeping” increased expression of gene response that persists
genes. Regulated genes, however, are expressed only indefinitely even after termination of the regulatory signal.
under certain conditions. They may be expressed in all
cells or in only a subset of cells, for example, hepato-
cytes. This ability to express certain genes in particular II. REGULATORY SEQUENCES AND
cells in eukaryotes is called tissue-specific expression. MOLECULES
The ability to regulate gene expression (that is, to deter-
mine if, how much, and when particular gene products Regulation of transcription, the initial step in all gene
will be made) gives the cell control over structure and expression, is controlled by regulatory sequences of DNA
function. It is the basis for cellular differentiation, morpho- that are usually embedded in the noncoding regions of the
genesis, and adaptability of any organism. Control of genome. The interaction between these DNA sequences

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II. Regulatory Sequences and Molecules 655

A Prokaryotes For most genes,

the main site
Trans-acting factors, usually
proteins, are synthesized from
of control is the genes that are different from the
DNA transcription of
genes targeted for regulation.
DNA to RNA.
Trans-acting factors bind to
cis-acting elements on DNA.
Transcription
mRNA
DNA
Translation
Protein
mRNA
mRN

B Eukaryotes
Trans-actin
Trans-acting
factor
Exons
DNA
Direction
of
transcripti
transcription
Introns
Pol II
Transcription DNA

Primary RNA transcript Transcribed

region
Cis-acting elements
mRNA isoforms

Cis-acting elements are DNA

sequences that are bound by trans-
acting regulatory factors.
Proteins

Modified proteins Figure 38.2 Cis-acting elements and trans-acting factors.

mRNA = messenger RNA; Pol II = RNA polymerase II.
In eukaryotes, gene expression is
also controlled at posttranscriptional
and posttranslational processes. A
C C C H
Zn Zn
Figure 38.1 Control of gene expression. mRNA = C C C H
messenger RNA.
Formation of zinc fingers in amino acid
and regulatory molecules, such as transcription factors, chain of protein.
can induce or repress the transcriptional machinery,
influencing the kinds and amounts of products that are B
produced. The regulatory DNA sequences are called cis-
acting because they influence expression of genes on
Amino
the same chromosome as the regulatory sequence (see acids of
p. 626). The regulatory molecules are called trans-acting DNA protein
because they can diffuse (transit) through the cell from with zinc
their site of synthesis to their DNA-binding sites (Fig. 38.2). finger
For example, a protein transcription factor (a trans-acting
Binding of protein to successive major
molecule) that regulates a gene on chromosome 6 might grooves of DNA by zinc finger motif.
itself have been produced from a gene on chromosome
11. The binding of proteins to DNA is through structural Figure 38.3 Zinc finger binding motif. A. Formation of zinc
motifs such as the zinc finger (Fig. 38.3), leucine zipper fingers in amino acid chain of protein. B. Binding of protein
(Fig. 38.4), or helix-turn-helix (Fig. 38.5) in the protein. to successive major grooves of DNA by zinc finger motif.
Motifs mediate the binding of regulatory proteins to DNA.
This binding to DNA is of high affinity, involving small binding can be understood by the example of cro protein
regions of protein and maintained by H-bonds, ionic binding to DNA. Cro has three antiparallel beta sheets and
interactions and van der Waals forces. Helix-turn-helix three alpha helices. Cro binds to DNA through alpha helix

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656 38. Regulation of Gene Expression

NH2 Protein 1

COOH

COOH
DNA binding Protein 2
domain NH2

Leucine zipper formation

by protein 1 and 2

Figure 38.4 Leucine zipper motif.

A. Messenger RNA transcription from

bacterial operons
In bacteria, the structural genes that encode proteins
DNA α3 involved in a particular metabolic pathway are often
α Helix 3 of cro protein found sequentially grouped on the chromosome along
binding to 5 bp. on with the cis-acting elements that regulate the tran-
major groove*
scription of these genes. The transcription product is
*All α helices and β sheets of cro protein a single polycistronic messenger RNA ([mRNA], see
not represented in diagram p. 621). The genes are, thus, coordinately regulated
(that is, turned on or off as a unit). This entire package
Figure 38.5 Helix-turn-helix motif. is referred to as an operon.

3 binding to 5 bp on major groove (Fig. 38.5). An example B. Operators in bacterial operons

of zinc finger binding is the binding of TF111A (a positive
regulator of 5S RNA gene transcription) to DNA. There is Bacterial operons contain an operator, a segment of
a zinc bound to cysteine and/or histidine 12–13 amino DNA that regulates the activity of the structural genes of
acids apart. The presence of this zinc causes ‘fingers” the operon by reversibly binding a protein known as the
to be made in the protein chain which now binds to 5 bp repressor. If the operator is not bound by the repressor,
in successive major grooves of DNA (Figs 38.3A and B). RNA polymerase (RNA pol) binds the promoter, passes
In the leucine zipper motif, proteins form an alpha helix over the operator, and reaches the protein-coding genes
with a periodic repeat of leucine residues at every 7th that it transcribes to mRNA. If the repressor is bound to
position. This makes a leucine line on one side of the the operator, the polymerase is blocked and does not
protein. These leucines on one side of the protein “zip” produce mRNA. As long as the repressor is bound to
with another such protein to form a DNA-binding region. the operator, no mRNA (and, therefore, no proteins) are
made. However, when an inducer molecule is present, it
An example of leucine zipper motif is Fos and Jun regu-
binds to the repressor, causing the repressor to change
latory proteins (Fig. 38.4).
shape so that it no longer binds the operator. When this
happens, RNA pol can initiate transcription. One of the
III. REGULATION OF PROKARYOTIC best-understood examples is the inducible lactose (lac)
operon of E. coli that illustrates both positive and nega-
GENE EXPRESSION
tive regulation (Fig. 38.6).

In prokaryotes such as the bacterium Escherichia coli

(E. coli), regulation of gene expression occurs primarily
C. Lactose operon
at the level of transcription and, in general, is mediated The lac operon contains the genes that code
by the binding of trans-acting proteins to cis-acting regu- for three proteins involved in the catabolism of
latory elements on their single DNA molecule (chromo- the disaccharide lactose: the lacZ gene codes for
some). [Note: Regulating the first step in the expression β-galactosidase, which hydrolyzes lactose to galac-
of a gene is an efficient approach, insofar as energy is not tose and glucose; the lacY gene codes for a permease,
wasted making unneeded gene products.] Transcriptional which facilitates the movement of lactose into the cell;
control in prokaryotes can involve the initiation or prema- and the lacA gene codes for thiogalactoside transacet-
ture termination of transcription. ylase, which acetylates lactose. [Note: The physiologic

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III. Regulation of Prokaryotic Gene Expression 657

A +– Lactose
Glucose
Operon repressed (off)

CAP pprotein lacl CAP

(unbound) gene site Operator
lacZ gene lacY gene lacA gene

Adenylyl cyclase
is inactive in the Promoter
presence of glucose,
and CAP is not mRNA
bound to cAMP:
catabolite repression.
Repressor Transcription is No mRNA and,
protein prevented by the therefore, no proteins
repressor protein. are produced.*

B –+ Glucose
Lactose Operon induced (on)
on
n) Operator is not blocked, and
the CAP site is occupied. RNA
polymerase can efficiently
initiate transcription.
lacl RNA polymerase
gene lacZ gene lacY gene lacA gene

CAP Operator
cAMP site Promoter
CAP mRNA
mRNA
b-Galactosidase Galactoside Thiogalactoside
permease transacetylase

Repressor Allolactose binds to a repressor

Allolactose protein, causing a conformational
Inactive repressor change that prevents the
repressor binding to the operator.
Adenylyl cyclase is active in the absence of glucose, producing
cAMP that binds to CAP. cAMP–CAP binds the CAP site.

C ++ Glucose
Lactose Operon uninduced Although the repressor is inactive, the
CAP-binding site is empty so RNA polymerase
cannot efficiently initiate transcription.

CAP lacl CAP

(unbound) gene site
ite Operator lacZ gene lacY gene lacA gene

Adenylyl cyclase
is inactive in the
presence of glucose, Promoter
and CAP is not
bound to cAMP: There are very low (basal) levels of mRNA and,
catabolite repression. therefore, protein expression.

Repressor

Allolactose Inactive repressor

Figure 38.6 The lactose operon of Escherichia coli in the presence of A. only glucose, B. only lactose, and C. both
sugars. *[Note: Even when the operon has been turned off, the repressor transiently dissociates from the operator at a
slow rate, allowing a very low level of expression. The synthesis of a few molecules of permease (and β-galactosidase)
allows the organism to respond rapidly should glucose become unavailable.] CAP = catabolite activator protein; cAMP
= cyclic adenosine monophosphate; mRNA = messenger RNA.

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658 38. Regulation of Gene Expression

function of this acetylation is unknown.] All of these known as catabolite repression) so no cAMP–CAP
proteins are maximally produced only when lactose is complex forms, and the CAP site remains empty.
available to the cell and glucose is not. [Note: Bacteria Therefore, the RNA pol is unable to effectively
use glucose, if available, as a fuel in preference to any initiate transcription, even though the repressor is
other sugar.] The regulatory portion of the operon is not bound to the O site. Consequently, the three
upstream of the three structural genes and consists structural genes of the operon are expressed only
of the promoter region where RNA pol binds and two at a very low (basal) level (see Fig. 38.6C). [Note:
additional sites, the operator (O) and the catabolite Induction causes a 50-fold enhancement over
activator protein (CAP) sites, where regulatory proteins basal expression.]
bind. The lacZ, lacY, and lacA genes are maximally
expressed only when the O site is empty and the CAP D. Tryptophan operon
site is bound by a complex of cyclic adenosine mono-
phosphate ([cAMP], see p. 190) and the CAP, some- The tryptophan (trp) operon contains five struc-
times called the cAMP regulatory protein (CRP). A tural genes that code for enzymes required for the
regulatory gene, the lacI gene, codes for the repressor synthesis of the amino acid tryptophan. As with the
protein (a trans-acting factor) that binds to the O site lac operon, the trp operon is subject to negative
with high affinity. [Note: The lacI gene has its own control. However, for the repressible trp operon, nega-
promoter and is not part of the lac operon.] tive control includes Trp itself binding to a repressor
protein and facilitating the binding of the repressor to
1. When only glucose is available: In this case, the the operator: Trp is a corepressor. Because repres-
lac operon is repressed (turned off). Repression sion by Trp is not always complete, the trp operon,
is mediated by the repressor protein binding unlike the lac operon, is also regulated by a process
via a helix-turn-helix motif (Fig. 38.5) to the O known as attenuation. With attenuation, transcription
site, which is downstream of the promoter (see is initiated but is terminated well before completion
Fig. 38.6A). Binding of the repressor interferes with (Fig. 38.7). If Trp is plentiful, transcription initiation that
the binding of RNA pol to the promoter, thereby escaped repression by Trp is attenuated (stopped) by
inhibiting transcription of the structural genes. the formation of an attenuator, a hairpin (stem-loop)
This is an example of negative regulation. structure in the mRNA similar to that seen in rho-
independent termination (see p. 624). [Note: Because
2. When only lactose is available: In this case, the
transcription and translation are temporally linked in
lac operon is induced (maximally expressed, or
prokaryotes (see p. 642), attenuation also results in
turned on). A small amount of lactose is converted
the formation of a truncated, nonfunctional peptide
to an isomer, allolactose. This compound is
product that is rapidly degraded.] If Trp becomes
an inducer that binds to the repressor protein,
scarce, the operon is expressed. The 5′-end of the
changing its conformation so that it can no longer
mRNA contains two adjacent codons for Trp. The
bind to the O site. In the absence of glucose,
lack of Trp causes ribosomes to stall at these codons,
adenylyl cyclase is active, and cAMP is made and
covering regions of the mRNA required for formation
binds to the CAP. The cAMP–CAP trans-acting
of the attenuation hairpin. This prevents attenuation
complex binds to the CAP site, causing RNA pol
and allows transcription to continue.
to initiate transcription with high efficiency at the
promoter site (see Fig. 38.6B). This is an example
of positive regulation. The transcript is a single Transcriptional attenuation can occur in prokaryotes
polycistronic mRNA molecule that contains three because translation of an mRNA begins before its
sets of start and stop codons. Translation of the synthesis is complete. This does not occur in eukary-
mRNA produces the three proteins that allow otes because the presence of a membrane-bound
lactose to be used for energy production by the nucleus spatially and temporally separates transcrip-
cell. [Note: In contrast to the inducible lacZ, lacY, tion and translation.
and lacA genes, whose expression is regulated,
the lacI gene is constitutive. Its gene product, the
repressor protein, is always made and is active E. The genetic switch of bacteriophage lambda
unless the inducer is present.]
The genetic switch of bacteriophage lambda
3. When both glucose and lactose are available: provides an example of two genes regulating each
In this case, the lac operon is uninduced, and other’s expression through protein-DNA inter-
transcription is negligible, even if lactose is actions. A bacteriophage infects a bacterium and
present at a high concentration. Adenylyl cyclase incorporates its genomic material inside the bacte-
is inhibited in the presence of glucose (a process rial genome. Now, if there is presence of nutrition

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IV. Regulation of Eukaryotic Gene Expression 659

Hairpin structure
Transcription Self-complementary sequences
terminator in certain regions of the mRNA
Nascent peptide cause a hairpin structure to form,
which attenuates (prematurely
terminates) transcription.

mRNA + “Terminated”
RNA polymerase

Ribosome translating mRNA Ribosome initiates translation of

mRNA as it is being transcribed.

Figure 38.7 Attenuation of transcription of the trp operon when tryptophan is plentiful. mRNA = messenger RNA.

Bacteriophage releasing
its DNA inside bacteria

Bacteria with its

circular DNA

Multiple
Unfavorable
replications of
conditions Favorable
Bacteriophage DNA
conditions

Lysogenic Lytic
pathway pathway

Bacteriophage Induction by
DNA incorporated UV light, nutrition New phage
and dormant in etc. reassembly
bacterial DNA

Phage DNA formation

and replication Release of newly
formed bacteriophages

Figure 38.8 Molecular switch of bacteriophage λ: lytic and lysogenic phase.

and sunlight, it can go into the lytic phase, replicate IV. REGULATION OF EUKARYOTIC
inside the bacteria and release its copies by lysing GENE EXPRESSION
the bacteria (Fig. 38.8). For this, it needs expression
of Cro gene (Fig. 38.9).
The higher degree of complexity of eukaryotic genomes,
The Cro protein will also suppress the repressor gene as well as the presence of a nuclear membrane, necessi-
by protein-DNA interactions. The lytic phase is a tates a wider range of regulatory processes. As with the
type C response which is irreversible. In case there prokaryotes, transcription is the primary site of regula-
is non-availability of nutrients and sunlight, the bacte- tion. Again, the theme of trans-acting factors binding to
riophage will infect the bacteria but lie dormant. The cis-acting elements is seen. Operons, however, are not
dormancy will be supported by activation of repressor found in eukaryotes, which must use alternate strate-
gene and suppression of Cro gene by protein-DNA gies to solve the problem of how to coordinately regulate
interactions. In this lysogenic phase, repressor protein all the genes required for a specific response. In eukary-
is made, which binds to the Cro promoter to suppress otes, gene expression is also regulated at multiple levels
synthesis of Cro protein. Lysogenic phase is reversible other than transcription. For example, the major modes
and can proceed to lytic phase in favorable conditions. of posttranscriptional regulation at the mRNA level are

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660 38. Regulation of Gene Expression

OR3 OR2 OR1

Gene for repressor Gene for Cro
Repressor Cro
promoter promoter
Repressor RNA Cro RNA
Maximum Maximum
affinity affinity

Repressor protein Cro protein

Lysogenic or
Lytic growth
prophage

Molecular switch of bacteriophage λ

Two genes regulating each other’s activity
In lysogenic cycle, the repressor gene is "ON" and repressor protein is
formed. This protein inhibits the expression of Cro gene.
In lytic cycle, the Cro gene is "ON" and repressor gene is inactive.

Figure 38.9 Molecular switch of bacteriophage λ: expression of cro gene.

alternative mRNA splicing and polyadenylation, control (UASGal). Binding of Gal4 to UASGal through zinc
of mRNA stability, and control of translational efficiency. fingers in its DBD occurs in both the absence and
Additional regulation at the protein level occurs by mech- presence of galactose. When the sugar is absent,
anisms that modulate stability, processing, or targeting of the regulatory protein Gal80 binds Gal4 at its TAD,
the protein. thereby inhibiting gene transcription (Fig. 38.10A).
When present, galactose activates the Gal3
A. Coordinate regulation protein. Gal3 binds Gal80, thereby allowing Gal4
to activate transcription (Fig. 38.10B). [Note:
The need to coordinately regulate a group of genes Glucose prevents the use of galactose by inhib-
to cause a particular response is of key importance in iting expression of Gal4 protein.]
organisms with more than one chromosome. An under-
lying theme occurs repeatedly: A trans-acting protein 2. Hormone response system: Hormone response
functions as a specific transcription factor (STF) that elements (HRE) are DNA sequences that bind trans-
binds to a cis-acting regulatory consensus sequence acting proteins and regulate gene expression in
(see p. 599) on each of the genes in the group even response to hormonal signals in multicellular
if they are on different chromosomes. [Note: The STF organisms. Hormones bind to either intracellular
has a DNA-binding domain (DBD) and a transcription (nuclear) receptors (for example, steroid hormones;
activation domain (TAD). The TAD recruits coactiva- see p. 360) or cell-surface receptors (for example,
tors, such as histone acetyltransferases (see p. 624), the peptide hormone glucagon; see p. 444).
and the general transcription factors (see p. 626) that,
along with RNA pol, are required for formation of a. Intracellular receptors: Members of the
the transcription initiation complex at the promoter. nuclear receptor superfamily, which includes
Although the TAD recruits a variety of proteins, the the steroid hormone (glucocorticoids, mineralo-
specific effect of any one of them is dependent upon corticoids, androgens, and estrogens), vitamin
the protein composition of the complex. This is known D, retinoic acid, and thyroid hormone recep-
as combinatorial control.] Examples of coordinate tors, function as STF. In addition to domains
regulation in eukaryotes include the galactose circuit for DNA-binding and transcriptional activa-
and the hormone response system. tion, these receptors also contain a ligand-
binding domain. For example, the steroid
1. Galactose circuit: This regulatory scheme allows hormone cortisol (a glucocorticoid) binds intra-
for the use of galactose when glucose is not avail- cellular receptors at the ligand-binding domain
able. In yeast, a unicellular organism, the genes (Fig. 38.11). Binding causes a conformational
required to metabolize galactose are on different change in the receptor that activates it. The
chromosomes. Coordinated expression is medi- receptor–hormone complex enters the nucleus,
ated by the protein Gal4 (Gal = galactose), a STF dimerizes, and binds via a zinc finger motif to
that binds to a short regulatory DNA sequence DNA at a regulatory element, the glucocorticoid
upstream of each of the genes. The sequence response element (GRE) that is an example of
is called the upstream activating sequence Gal a hormone response element (HRE). Binding

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IV. Regulation of Eukaryotic Gene Expression 661

A – Galactose Circuit off

TAD of GAL4 is
Gal80 blocked by Gal80.
TAD Transcription
Gal4
DBD

UASGal Promoter Target gene

Zinc finger

B + Galactose Circuit on
TAD of GAL4 can recruit the proteins
needed for transcription initiation.
Gal80

Gal3 TAD
Gal4 Transcription
DBD mRNA

UASGal Promoter Target gene

Figure 38.10 Regulation of galactose (gal) circuit in yeast in the A. absence and B. presence of galactose. [Note: Target
genes, whether on the same or a different chromosome, each have an upstream activating sequence galactose (UASGal).]
TAD = transcription activation domain; DBD = DNA-binding domain; mRNA = messenger RNA.

GRE
Cortisol
DNA
GR
GR GR
GR GR The hormone–receptor complex
GR
Coactivators 2 interacts with specific regulatory
GR dimer DNA sequences such as GRE.
Basal
transcription RNA polymerase II
complex
The binding of a steroid
1 hormone to its nuclear
Start of transcription
receptor causes a TATA box
conformational change
in the receptor that The hormone–receptor complex
uncovers its zinc finger Regulated 3 in association with coactivator
DNA-binding domain. gene proteins controls the transcrip-
transcription tion of targeted genes.

Figure 38.11 Transcriptional regulation by intracellular steroid hormone receptors. GRE = glucocorticoid response
element; GR = glucocorticoid receptor.

allows recruitment of coactivators to the TAD b. Cell-surface receptors: These receptors

and results in expression of cortisol-responsive include those for insulin, epinephrine, and
genes, each of which is under the control of glucagon. Glucagon, for example, is a peptide
its own GRE. Binding of the receptor–hormone hormone that binds its G protein–coupled
complex to the GRE allows coordinate expres- plasma membrane receptor on glucagon-
sion of a group of target genes, even though responsive cells. This extracellular signal
these genes are on different chromosomes. is then transduced to intracellular cAMP,
The GRE can be located upstream or down- a second messenger (Fig. 38.12; also see
stream of the genes it regulates and at great Fig. 13.7 on p. 190), which can affect protein
distances from them. The GRE, then, can func- expression (and activity) through protein kinase
tion as a true enhancer (see p. 627). [Note: If A–mediated phosphorylation. In response to
associated with repressors, hormone–receptor a rise in cAMP, a trans-acting factor (cAMP
complexes inhibit transcription.] response element–binding [CREB] protein) is

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662 38. Regulation of Gene Expression

B. Messenger RNA processing and use

Hormone or extracellular
signal molecule Eukaryotic mRNA undergoes several processing
Cell membrane events before it is exported from the nucleus to the
cytoplasm for use in protein synthesis. Capping at
the 5′-end (see p. 628), polyadenylation at the 3′-end
Receptor
(see p. 629), and splicing (see p. 629) are essential for
the production of a functional eukaryotic messenger
Adenylyl from most pre-mRNA. Variations in splicing and poly-
cyclase adenylation can affect gene expression. In addition,
cAMP ATP
messenger stability also affects gene expression.

1. Alternative splicing: Tissue-specific protein

isoforms can be made from the same pre-mRNA
When CREB is phosphorylated,
it can bind to CRE and activate through alternative splicing, which can involve exon
the transcription machinery. skipping (loss), intron retention, and use of alter-
native splice-donor or -acceptor sites (Fig. 38.13).
CREB Transcription factors For example, the pre-mRNA for tropomyosin (TM)
undergoes tissue-specific alternative splicing to
RNA yield a number of TM isoforms (see p. 630). [Note:
P polymerase II
Over 90% of all human genes undergo alternative
splicing.]

2. Alternative polyadenylation: Some pre-mRNA

CRE
transcripts have more than one site for cleavage
Start of transcription and polyadenylation. Alternative polyadenylation
(APA) generates mRNA with different 3′-ends,
Figure 38.12 Transcriptional regulation by receptors altering the untranslated region (UTR) or the coding
located in the cell membrane. [Note: Cyclic adenosine (translated) sequence. [Note: APA is involved in the
monophosphate (cAMP) activates protein kinase A that production of the membrane-bound and secreted
phosphorylates cAMP response element–binding (CREB) forms of immunoglobulin M.]
protein.] CRE = cAMP response element.
The use of alternative splicing and polyadenylation
phosphorylated and activated. Active CREB
sites, as well as alternative transcription start sites
protein binds via a leucine zipper motif to explains, at least in part, how the ~20,000 to 25,000
a cis-acting regulatory element, the cAMP genes in the human genome can give rise to well over
response element (CRE), resulting in tran- 100,000 proteins.
scription of target genes with CRE in their
promoters. [Note: The genes for phosphoenol-
pyruvate carboxykinase and glucose 6-phos- 3. Messenger RNA editing: Even after mRNA has
phatase, key enzymes of gluconeogenesis been fully processed, it may undergo an additional
(see p. 215), are examples of genes upregu- posttranscriptional modification in which a base
lated by the cAMP/CRE/CREB system.] in the mRNA is altered. This is known as RNA

mRNA
Gene
Translation
1 2 3 4 5
Exon Exon Exon Exon Exon

1 2 3 4 5
Alternative
Splicing
DNA Protein A
1 2 4 5 Translation
mRNA

Protein B

Figure 38.13 Tissue-specific alternative splicing produces different proteins, or isoforms, from a single gene. mRNA =
messenger RNA.

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IV. Regulation of Eukaryotic Gene Expression 663

message) being made in the intestine (and incor-

pre-mRNA porated into chylomicrons) than is made in the liver
AUG CAA UUA (apo B-100, full-length, incorporated into VLDL).
5ʹ 3ʹ
(Gln)
4. Messenger RNA stability: How long an mRNA
Splicing remains in the cytosol before it is degraded influ-
Polyadenylation
Editing: C gets ences how much protein product can be produced
deaminated to U from it. Regulation of iron metabolism and the
gene-silencing process of RNA interference (RNAi)
mRNA
illustrate the importance of mRNA stability in the
AUG UAA UUA
5ʹ 3ʹ regulation of gene expression.
(stop)
a. Iron metabolism: Transferrin (Tf) is a plasma
Translation protein that transports iron. Tf binds to cell-
apo B-48 surface receptors (transferrin receptors [TfR])
protein that get internalized and provide cells, such
as erythroblasts, with iron. The mRNA for
COO–
+
NH3
the TfR has several cis-acting iron-respon-
sive elements (IRE) in its 3′-UTR. IRE have a
Figure 38.14 Editing of apolipoprotein (apo) B pre- short stem-loop structure that can be bound
mRNA in the intestine and generation of the apo B-48 by trans-acting iron regulatory proteins (IRP),
protein needed for chylomicron synthesis. Gln = glutamine; as shown in Figure 38.15. When the iron
mRNA = messenger RNA; A = adenine; C = cytosine; G = concentration in the cell is low, the IRP bind
guanine; U = uracil. to the 3′-IRE and stabilize the mRNA for TfR,
allowing TfR synthesis. When intracellular iron
editing. An important example in humans occurs levels are high, the IRP dissociate. The lack of
with the transcript for apolipoprotein (apo) B, an IRP bound to the mRNA hastens its destruc-
essential component of chylomicrons (see p. 350) tion, resulting in decreased TfR synthesis.
and very-low-density lipoproteins ([VLDL], see p. [Note: The mRNA for ferritin, an intracellular
352). Apo B mRNA is made in the liver and the protein of iron storage, has a single IRE in its
small intestine. However, in the intestine only, the 5′-UTR. When iron levels in the cell are low,
cytosine (C) base in the CAA codon for glutamine IRP bind the 5′-IRE and prevent the use of the
is enzymatically deaminated to uracil (U), changing mRNA, and less ferritin is made. When iron
the sense codon to the nonsense or stop codon accumulates in the cell, the IRP dissociate,
UAA, as shown in Figure 38.14. This results in a allowing synthesis of ferritin molecules to store
shorter protein (apo B-48, representing 48% of the the excess iron. Aminolevulinic acid synthase

3ʹ-Iron-responsive element (IRE)

7-CH3-G (A)n
TfR mRNA 5ʹ Coding 3ʹ

Low iron: IRP binds IRE High iron: IRP does not bind
Iron regulatory protein (IRP)

IRP on IRE
Unbound IRE

5ʹ Coding 3ʹ 5ʹ Coding 3ʹ
TfR mRNA is stabilized and TfR mRNA
used in protein synthesis is degraded

TfR made No TfR made

Figure 38.15 Regulation of transferrin receptor (TfR) synthesis. [Note: The IRE are located in the 3ʹ-UTR (untranslated
region) of TfR messenger RNA (mRNA).] 7-CH3-G = 7-methylguanosine cap; (A)n = polyadenylate tail.

LIR Biochemistry.indb 663 20-05-2024 03:18:09

664 38. Regulation of Gene Expression

2, the regulated enzyme of heme synthesis

DNA
(see p. 416) in erythroblasts, also contains a
5′-IRE.] (See Chapter 33 for a discussion of
RNA polymerase II
iron metabolism.)
5ʹ-Cap
b. RNA interference: RNAi is a mechanism of Pri-miRNA
gene silencing through decreased expres- 3ʹ-Tail
Drosha
sion of mRNA, either by repression of trans-
lation or by increased degradation. It plays a Pre-miRNA
key role in such fundamental processes as cell NUCLEUS
proliferation, differentiation, and apoptosis.
RNAi is mediated by short (~22 nucleotides),
CYTOPLASM Dicer
noncoding RNA called microRNA (miRNA).
The miRNA arise from far longer, genomically miRNA
encoded nuclear transcripts, primary miRNA
Guide strand of double-
(pri-miRNA), that are partially processed in the stranded miRNA
nucleus to pre-miRNA by an endonuclease associates with RISC
(Drosha) then transported to the cytoplasm. and hybridizes with
target mRNA
There, an endonuclease (Dicer) completes
Target
the processing and generates short, double- mRNA
stranded miRNA. A single strand (the guide
or antisense strand) of the miRNA associates RISC Guide strand
with a cytosolic protein complex known as the
RNA-induced silencing complex (RISC). The
guide strand hybridizes with a complemen-
Translational Degradation of
tary sequence in the 3′-UTR of a full-length repression of target mRNA by
target mRNA, bringing RISC to the mRNA. target mRNA Argonaute/Ago/Slicer
This can result in repression of translation of
the mRNA or its degradation by an endonu- Figure 38.16 Biogenesis and actions of microRNA
clease (Argonaute/Ago/Slicer) of the RISC. (miRNA). [Note: The extent of complementarity between the
The extent of complementarity appears to be target messenger RNA (mRNA) and the miRNA determines
the determining factor (Fig. 38.16). RNAi can the final outcome, with perfect complementarity resulting
also be triggered by the introduction of exog- in mRNA degradation.] Pri = primary; RISC = RNA-induced
enous double-stranded short interfering RNA silencing complex.
(siRNA) into a cell, a process that has enor-
mous therapeutic potential. 5. Messenger RNA translation: Regulation of gene
expression can also occur at the level of mRNA
i. RNA interference–based therapeu- translation. One mechanism by which transla-
tics: The first clinical trial of RNAi-based tion is regulated is through phosphorylation of
therapy involved the neovascular form of the eukaryotic translation initiation factor, eIF-2
age-related macular degeneration (AMD), (Fig. 38.17). Phosphorylation of eIF-2 inhibits its
which is triggered by overproduction of function and so inhibits translation at the initiation
vascular endothelial growth factor (VEGF), step (see p. 644). [Note: Phosphorylation of eIF-2
leading to the sprouting of excess blood prevents its reactivation by inhibiting GDP-GTP
vessels behind the retina. The vessels exchange.] Phosphorylation is catalyzed by
leak, clouding and often entirely destroying kinases that are activated in response to environ-
vision (therefore, neovascular AMD is also mental conditions, such as amino acid starvation,
referred to as wet AMD). An siRNA was heme deficiency in erythroblasts, the presence of
designed to target the mRNA of VEGF and double-stranded RNA (signaling viral infection),
promote its degradation. Although consid- and the accumulation of misfolded proteins in the
erable effort and resources have been rough endoplasmic reticulum (see p. 649).
expended to develop RNAi-based ther-
apeutics, especially for the treatment of
cancer, no products have gone from trials
C. Regulation through variations in DNA
to the market. The research applications Gene expression in eukaryotes is also influenced
of RNAi, however, have grown rapidly. by the accessibility of DNA to the transcriptional

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IV. Regulation of Eukaryotic Gene Expression 665

Amino acid starvation, heme deficiency, NH2 NH2

accumulation of misfolded proteins CH3
in RER, double-stranded RNA N Methyltransferase N

+ O N H SAM O N H
SAH
Kinase
eIF-2 eIF2- P Cytosine 5-Methylcytosine
+ ATP + ADP
Phosphatase
Figure 38.18 The methylation of cytosine in
Pi
eukaryotic DNA. SAM = S-adenosylmethionine; SAH =
Figure 38.17 Regulation of translation initiation in S-adenosylhomocysteine
eukaryotes by phosphorylation of eukaryotic translation
initiation factor, eIF-2. RER = rough endoplasmic reticulum; of gene product produced. An increase in copy
ADP = adenosine diphosphate; Pi = inorganic phosphate; number (gene amplification) has contributed to
P = phosphate.
increased genomic complexity and is still a normal
developmental process, e.g., ribosomal protein
apparatus, the amount of DNA, and the arrangement genes amplify as a new cell is being made. Gene
of DNA. [Note: Localized transitions between the B amplification is also seen in some diseases and
and Z forms of DNA (see p. 598) can also affect gene in response to particular chemotherapeutic drugs
expression.] such as methotrexate, an inhibitor of the enzyme
dihydrofolate reductase (DHFR), required for the
1. Access to DNA: In eukaryotes, DNA is found synthesis of thymidine triphosphate (TTP) in the
complexed with histone and nonhistone proteins pyrimidine biosynthetic pathway (see p. 587). TTP
to form chromatin (see p. 608). Transcriptionally is essential for DNA synthesis. Gene amplifica-
active, decondensed chromatin (euchromatin) tion results in an increase in the number of DHFR
differs from the more condensed, inactive form genes and resistance to the drug, allowing TTP to
(heterochromatin) in a number of ways. Active be made.
chromatin contains histone proteins that have
been covalently modified at their amino terminal 3. Arrangement of DNA: The process by which
ends by reversible methylation, acetylation, or immunoglobulins (antibodies) are produced
phosphorylation (see p. 625 for a discussion of by B lymphocytes involves permanent rear-
histone acetylation/deacetylation by histone acet- rangements of the DNA in these cells. The
yltransferase and histone deacetylase). Such modi- immunoglobulins (for example, IgG) consist of
fications decrease the positive charge of these two light and two heavy chains, with each chain
basic proteins, thereby decreasing the strength containing regions of variable and constant amino
of their association with negatively charged DNA. acid sequence. The variable region is the result
This relaxes the nucleosome (see p. 609), allowing of somatic recombination of segments within
transcription factors access to specific regions on both the light- and the heavy-chain genes. During
the DNA. Nucleosomes can also be repositioned, B-lymphocyte development, single variable (V),
an ATP-requiring process that is part of chro- diversity (D), and joining (J) gene segments are
matin remodeling. Another difference between brought together through gene rearrangement
transcriptionally active and inactive chromatin to form a unique variable region (Fig. 38.19).
is the extent of methylation of cytosine bases in This process allows the generation of 109−1011
CG-rich regions (CpG islands) in the promoter different immunoglobulins from a single gene,
region of many genes. Methylation is by methyl- providing the diversity needed for the recogni-
transferases that use S-adenosylmethionine as the tion of an enormous number of antigens. [Note:
methyl donor (Fig. 38.18). Transcriptionally active Pathologic DNA rearrangement is seen with trans-
genes are less methylated (hypomethylated) location, a process by which two different chro-
than their inactive counterparts, suggesting that mosomes exchange DNA segments.]
DNA hypermethylation silences gene expression.
Modification of histones and methylation of DNA 4. Mobile DNA elements: Transposons (Tn) are
are epigenetic in that they are heritable changes mobile segments of DNA that move in an essen-
in DNA that alter gene expression without altering tially random manner from one site to another on
the base sequence. the same or a different chromosome. Movement
is mediated by transposase, an enzyme encoded
2. Amount of DNA: A change up or down in the by the Tn itself. Movement can be direct, in which
number of copies of a gene can affect the amount transposase cuts out and then inserts the Tn at a

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666 38. Regulation of Gene Expression

new site, or replicative, in which the Tn is copied

V J C and the copy inserted elsewhere while the original
DNA
germline remains in place. In eukaryotes, including humans,
replicative transposition frequently involves an
RNA intermediate made by a reverse transcrip-
Re-arranged DNA tase (see p. 608), in which case the Tn is called
VL CL a retrotransposon. Transposition has contributed
mRNA to structural variation in the genome but also has
the potential to alter gene expression and even to
Antibody
cause disease. Tn comprise ~50% of the human
genome, with retrotransposons accounting for
Antigen binding 90% of Tn. Although the vast majority of these
site retrotransposons have lost the ability to move,
some are still active. Their transposition is thought
to be the basis for some rare cases of hemophilia
Note: The immunoglobulin genes are comprised of numerous A and Duchenne muscular dystrophy. [Note: The
discontinuous segments in the DNA. growing problem of antibiotic-resistant bacteria is
As B cells develop, these DNA segments are chosen and joint
together such that each mature B cell and plasma cell has a
a consequence, at least in part, of the exchange
unique rearrangement profile for a unique antibody of plasmids among bacterial cells. If the plasmids
contain Tn-carrying antibiotic resistance genes,
Figure 38.19 Immunoglobulin gene rearrangement in the recipient bacteria gain resistance to one or
antibody-producing cell. more antimicrobial drugs.]

Additional interesting features about eukaryotic gene expression

• In eukaryotes, there can be further binding of multiple proteins which lead to extra DNA bends and approximations
that exponentially increase rate of transcription. This formation of enhanceosomes helps the eukaryotes to deal
with an urgent and excessive need like increased transcription of human beta interferon gene in response to human
immunodeficiency virus (HIV) infection.
• Locus control regions (LCRs) are segments of DNA that controls gene clusters which may be near or far apart
from each other (but on the same chromosome). This control is a level above regulated expression of genes and
may be related to chromatin structure. For example, an LCR controls expression of globin gene family over a large
region of DNA. Insulators are segments of DNA beyond which the influence of LCR will not extend.
• Gene switching is a phenomenon in which transcription and translation of a given gene stops and a related one is
turned on. This is seen in development of hemoglobin from embryonic to adult life and in immunoglobin response
to an antigen (Chapter 43).
• Eukaryotes have tissues and therefore their differentiation requires some specific processes. Tissue-specific expres-
sion implies the production of particular products in particular tissues only, e.g., production of insulin from pancreas
and immunoglobulins from B-lymphocytes. Some ways in which tissue-specific expression can be achieved include
enhancer or enhancer-like elements which can act in a tissue-specific manner to regulate gene expression.
• Genomic imprinting is the inheritance of some genes in a silent state from one parent (imprinted by methylation
which inhibits its expression), while the other parental gene is expressed. Further, eukaryotes also have the ability
of an SOS response which is the coordinated regulation of unrelated genes having a common goal. For example,
when there is severe DNA damage, the common goal of limiting damage and preventing transmission to daughter
cells leads to coordination of DNA-repairing genes and cell division repression genes. Gene loss may also occur in
eukaryotes. It is the loss of genetic material from cells, e.g., RBC which are without nucleus.
• The meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA, are another
challenge in understanding eukaryote gene expression. These epigenetic modifications have been targeted in
drug therapies.
• Gene silencing is also one of the epigenetic process of gene regulation. It is the switching off a gene by mecha-
nisms other than genetic modifications. Common ways are transcriptional gene silencing by histone modifications,
DNA methylations and silencing by interfering RNA. This can be developmental as a part of tissue-specific expres-
sion and could also be a protection against transposons or viruses.

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V. Medical Perspectives 667

V. Medical Perspectives
• A resistance to methotrexate (MTX), an anticancer drug is often seen in patients receiving MTX for a long time. This is
because of gene amplification of dihydrofolate reductase (DHFR) gene leading to increased number of DHFR genes,
causing increased production of DHFR. Therefore, the clinician often needs to increase dosage of MTX in such patients
• As the individual grows from the embryonic stage to an adult, his hemoglobin changes to a different type. This is achieved
by gene switching. Embryonic hemoglobin has two alpha and two epsilon chains. This is replaced by fetal Hb at 6
months of life which has two alpha and two gamma chains. After birth, two alpha and two beta chains (HbA1) or two
alpha and two delta chains are synthesized (HbA2). Defects in gene switching can give rise to severe hemoglobinopathies.
• Gene switching is also responsible for class switching of immunoglobulins during transition from primary to secondary
immune response. During primary response IgM is produced, but in secondary response IgG with same antigen
specificity is synthesized.
• Therefore, to assess a recent infection, IgM antibodies are tested, and IgG antibodies are tested to assess an infec-
tion which happened some time ago.
• The inhibition of specific epigenetic enzymes can reverse the incorporation of an epigenetic mutation making these
enzymes attractive targets for cancer therapy. Drugs which inhibit DNA dimethyltransferase 1 (Azacytidine and Decitabine)
are approved for treatment of adult myeloid leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic
syndrome (MDS). Vorinostat is a histone deacetylase inhibitor used to treat cutaneous T-cell lymphoma (CTCL).
• Prader-Willi syndrome (PWS) and its sister syndrome, Angelman syndrome (AS), are classical examples of genomic
imprinting in humans. Both syndromes are associated with defects in chromosome 15. Paternal inheritance of a deletion
of this region is associated with PWS (characterized by hypotonia, obesity, and hypogonadism), while maternal inheritance
of the same deletion is associated with AS (characterized by epilepsy, tremors, and a perpetually smiling facial expression).

VI. Chapter Summary

• Gene expression results in the production of a functional gene product (either RNA or protein) through the processes
of transcription and translation (Fig. 38.20).
• Genes can be either constitutive (always expressed, housekeeping genes) or regulated (expressed only under certain
conditions in all cells or in a subset of cells). The ability to appropriately induce (positively regulate) or repress (nega-
tively regulate) genes is essential in all organisms. Regulation of gene expression occurs primarily at transcription
in both prokaryotes and eukaryotes and is mediated through trans-acting proteins binding to cis-acting regulatory
DNA elements. I
• n eukaryotes, regulation also occurs through DNA modifications and through posttranscriptional and posttransla-
tional processing. In prokaryotes, such as Escherichia coli, the coordinate regulation of genes whose protein prod-
ucts are required for a particular process is achieved through operons (groups of genes sequentially arranged on the
chromosome along with the regulatory elements that determine their transcription).
• The lac operon contains the Z, Y, and A structural genes, the protein products of which are needed for the catabolism
of lactose. It is subject to negative and positive regulation. When glucose is available, the operon is repressed by the
binding of the repressor protein (the product of the lacI gene) to the operator, thus preventing transcription. When
only lactose is present, the operon is induced by an isomer of lactose (allolactose) that binds the repressor protein,
preventing it from binding to the operator. In addition, cyclic adenosine monophosphate (cAMP) binds the catabo-
lite activator protein (CAP), and the complex binds the DNA at the CAP site. This increases promoter efficiency and
results in the expression of the structural genes through the production of a polycistronic messenger RNA (mRNA).
When both glucose and lactose are present, glucose prevents formation of cAMP, and transcription of these genes
is negligible.
• The trp operon contains genes needed for the synthesis of tryptophan (Trp), and, like the lac operon, it is regulated by
negative control. Unlike the lac operon, it is also regulated by attenuation, in which mRNA synthesis that escaped
repression by Trp is terminated before completion. Transcription of ribosomal RNA and transfer RNA is selectively
inhibited in prokaryotes by the stringent response to amino acid starvation.
• Translation is also a site of prokaryotic gene regulation: Excess ribosomal proteins bind the Shine-Dalgarno sequence
on their own polycistronic mRNA, preventing ribosomes from binding.
• Gene regulation is more complex in eukaryotes. Operons are not present, but coordinate regulation of the transcrip-
tion of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-
acting elements as seen in the galactose circuit in unicellular yeast.

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668 38. Regulation of Gene Expression

• In multicellular organisms, hormones can cause coordinated regulation, either through the binding of the hormone
receptor–hormone complex to the DNA (as with steroid hormones) or through the binding of a protein that is acti-
vated in response to a second messenger (as with glucagon). In each case, binding to DNA is mediated through
structural motifs such as the zinc finger. Co- and posttranscriptional regulation is also seen in eukaryotes and
includes alternative mRNA splicing and polyadenylation, mRNA editing, and variations in mRNA stability as seen
with transferrin receptor synthesis and with RNA interference. Regulation at the translational level can be caused
by the phosphorylation and inhibition of eukaryotic initiation factor-2.
• Gene expression in eukaryotes is also influenced by accessibility of DNA to the transcriptional apparatus (as seen with
epigenetic changes to histone proteins), the amount of DNA, and the arrangement of the DNA.

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VI. Chapter Summary 669

Regulation of Gene Expression: Prokaryotes

occurs primarily at also occurs at

Transcription Translation
through through

Operons Operons
encoding encoding

r-Proteins
regulated by
Enzymes for Enzymes for
use of lactose synthesis of Trp rRNA, tRNA
Specific r-protein
regulated by binding to SD
also regulated by sequence on
ppGpp its mRNA
Attenuation through resulting in

Stringent Inhibition of
regulated by response r-protein synthesis

Positive (inducer) Negative (Trp

and negative corepressor)
(repressor) control control

Regulation of Gene Expression: Eukaryotes

occurs primarily at also occurs at also occurs at also occurs at

Transcription Co- and post- Translation DNA level

transcription
through events through
through
Trans-acting proteins Phosphorylation
binding to cis- Variation in mRNA of eIF-2
acting elements processing and through
on the DNA stability seen with
through
as exemplified by
Amino acid starvation,
DNA binding motifs Alternative splicing heme deficiency,
such as zinc fingers and polyadenylation, dsRNA, unfolded
as exemplified by mRNA editing, protein in ER
RNAi
Gal4 protein binding
to UASGal

and by

Steroid hormone–
receptor complex Changes in access Changes in Changes in
binding to GRE to DNA amount of DNA arrangement of DNA
as exemplified by as exemplified by as exemplified by

Histone modification,
DNA methylation, Gene Immunoglobins,
and nucleosome amplification transposons
repositioning

Figure 38.20 Summary of key concepts for the regulation of gene expression. Trp = tryptophan; rRNA, tRNA, mRNA =
ribosomal, transfer, and messenger RNA, respectively; ppGpp; guanosine tetraphosphate; r-protein = ribosomal protein;
SD = Shine-Dalgarno; Gal = galactose; UAS = upstream activating sequence; GRE = glucocorticoid response element;
RNAi = RNA interference; eIF = eukaryotic initiation factor; ds = double stranded; ER = endoplasmic reticulum.

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