Genetic Regulation
OVERVIEW OF GENETIC REGULATION
• Regulation of gene expression is an essential feature in
maintaining the functional integrity of a cell.
• Increasing or decreasing the expression of a gene can occur
through a variety of mechanisms, but many of the important
ones involve regulating the rate of transcription.
• In addition to the basic transcription proteins, RNA
polymerase, sigma (prokaryotes), and TFIID (eukaryotes),
activator and repressor proteins help control the rate of the
process. These regulatory proteins bind to specific DNA
sequences associated with both prokaryotic and eukaryotic
gene regions.
• Other mechanisms are important, and, especially in
eukaryotes, gene expression is controlled at multiple levels.
REGULATION OF PROKARYOTIC GENE
EXPRESSION
• Regulation of gene expression in prokaryotes usually
involves either initiation or termination of transcription.
In bacteria, genes are often organized into operons.
• An operon is a set of structural genes coding for a
group of proteins required for a particular metabolic
function along with the regulatory region(s) that controls
the expression of the structural genes.
• The regulatory region is upstream (to the 5' side)
from the structural genes and coordinates their
regulation. An operon usually produces a
polycistronic mRNA that carries the information
for synthesis of all of the enzymes encoded by the
structural genes.
Two examples of transcriptional control in
prokaryotes are discussed:
• Regulation by activator and repressor proteins in
the lactose operon
• Attenuation control in the histidine operon
The lactose (lac) Operon
• The lactose operon is a portion of the bacterial
chromosome that controls the synthesis of three
enzymes involved in the metabolism of the sugar
lactose.
• Most bacteria carry out glycolysis, a pathway that
allows glucose to be metabolized as a carbon and
energy source.
• If glucose is unavailable, they can metabolize
alternative carbohydrates, but require proteins
(enzymes) in addition to those in glycolysis to do so.
• Lactose, a disaccharide of galactose and glucose,
represents one alternative sugar, and the genes of the
lactose operon encode the additional proteins
required for its metabolism.
• The cell expresses these genes only when lactose is
available and glucose is not. The structural genes of the
lactose operon include:
• The Z gene, which encodes a β-galactosidase (a
prokaryotic lactase)
• The Y gene, which encodes a galactoside permease, the
transport protein required for entry of lactose into the cell
• The A gene, which encodes a thiogalactoside
transacetylase enzyme that is not essential for lactose
metabolism and whose function is uncertain
• In addition, the i gene, which encodes the lac repressor
protein, is also considered part of the operon although it
is located at a distant site in the DNA. The i gene is
constitutively expressed (not regulated); thus, copies of
the lac repressor protein are always in the cell.
• Two gene regulatory proteins control the expression of
the lac operon:
• The lac repressor (encoded by the i gene), which binds to
a DNA sequence called the operator
• A cAMP-dependent activator protein, CAP, which binds
to a DNA sequence called the CAP site
The lactose
metabolism
Glucose and lactose control the expression by
different mechanisms:
• Lactose (or allolactose) induces gene expression
by preventing the repressor protein binding to
the operator sequence.
• Glucose represses gene expression by lowering
the level of cAMP in the cell, thus preventing
the cAMP-dependent activator binding to the
CAP-site sequence.
Coordinated Control of the Lactose Operon by Glucose
and Lactose
• Full expression of the lactose operon requires that both
mechanisms favor gene expression.
• The repressor protein must not bind at the operator, and
• The cAMP-dependent activator protein must bind to the
CAP site.
This in turn requires that
• Lactose is present (prevents repressor binding)
• Glucose is low (to allow cAMP to increase)
• Although intermediate levels of gene expression may
be possible in the cell, it is convenient to simplify the
situation in the following way:
• The only condition that allows high gene expression
is: lactose present, glucose absent.
• All other combinations of these sugars result in lowlevel expression.
Attenuation in the Histidine Operon
• The histidine operon encodes the enzymes of the
histidine biosynthetic pathway. It is advantageous to
the cell to produce these enzymes when histidine is
not available in the surroundings, but to turn off
their synthesis when histidine is readily available.
• The histidine operon and several other operons for
amino acid biosynthesis (e.g., tryptophan, leucine,
and phenylalanine) are regulated by premature
termination of transcription, a process known as
attenuation.
• Attenuation depends on the fact that transcription and
translation occur simultaneously in bacteria.
• In this process, transcription is constitutively
initiated, and the 5' untranslated region (UTR) of the
mRNA is followed by the coding region for a short
(non-functional) peptide (the leader peptide).
• If histidine is available in the growth medium,
transcription is terminated before RNA polymerase
reaches the structural genes of the operon
• This form of regulation is dependent on the speed of
the ribosome and the formation of two alternative
secondary structures in the mRNA molecule.
Attenuation Control of Transcription in the Histidine Operon
At High Levels of Histidine
• As soon as the Shine-Dalgarno sequence associated with
the leader peptide coding region appears in the 5' UTR of
the mRNA, a ribosome binds and begins translating the
message.
• The ribosome can move quickly because it can easily
find histidine to incorporate when it encounters histidine
codons in the mRNA.
• This allows the message to fold into a rho-independent
terminator of transcription (stem and loop + poly-U).
• RNA polymerase stops transcription before it reaches the
structural genes, and no enzymes are produced.
At Low Levels of Histidine
• A ribosome begins to synthesize the leader peptide, but
stalls at the histidine codons because it cannot readily
find histidine.
• Because the ribosome is covering up a different part of
the mRNA, the message will not fold into the correct
terminator structure, and RNA polymerase continues
transcription through the structural genes of the operon.
• Translation of the message produces all the enzymes of
the histidine biosynthetic pathway.
Note: Attenuation is not used as a regulatory mechanism in
eukaryotes, because transcription and translation are
independent events and occur in different subcellular
locations.
REGULATION OF EUKARYOTIC GENE
EXPRESSION
• In eukaryotic cells, DNA is packaged in chromatin
structures, and gene expression typically requires
activation to occur. Chromatin-modifying activities
include:
• Histone acetylases (favor gene expression) and
deacetylases (favor inactive chromatin)
• Scaffolding proteins that condense regions of the
chromatin (favor inactive chromatin)
• DNA methylating enzymes (favor inactive chromatin)
• Activator proteins (and a few repressors) are
important in eukaryotes, as they are in prokaryotes.
• The DNA sequences to which activator proteins
bind in eukaryotic DNA are called response
elements.
• A few response elements are located within the
promoter region (upstream promoter elements
[UPE]), but most are outside the promoter and
often clustered to form an enhancer region that
allows control of gene expression by multiple
signals.
Enhancers and Upstream Promoter Elements
Upstream Promoter Elements
• Only the proximity of the upstream promoter element to the 25 sequence distinguishes it from an enhancer.
Proximal promoter elements include:
• A CCAAT box (around -75) that binds a transcription factor NF-l (CTF)
• A GC-rich sequence that binds a general transcription factor SP-l
Enhancers
• Enhancers in the DNA are binding sites for activator
proteins. Enhancers have the following
characteristics:
– They may be up to 1000 base pairs away from the gene.
– They may be located upstream, downstream, or within an
intron of the gene they control.
– The orientation of the enhancer sequence with respect to
the gene is not important.
– Enhancers can appear to act in a tissue-specific manner if
the DNA-binding proteins that interact with them are
present only in certain tissues.
– Enhancers may be brought close to the basal promoter
region in space by bending of the DNA molecule.
Stimulation of Transcription by an
Enhancer and Its Associated Transcription Factors
• Similar sequences that bind repressor proteins in eukaryotes
are called silencers. There are fewer examples of these
sequences known, and the mechanisms through which they act
are not clear.
Note
• The Ig heavy chain locus has an enhancer in the large intron
separating the coding regions for the variable domain from the
coding regions for the constant domains
Note
Cis and Trans Regulatory Elements
• The DNA regulatory base sequences (e.g., promoters,
enhancers, response elements, and UPEs) in the vicinity of
genes that serve as binding sites for proteins are often called
"cis" regulators.
• Transcription factors (and the genes that code for them) are
called "trans" regulators. Trans regulatory protein can diffuse
through the cell to their point of action.
Transcription Factors
• The activator proteins that bind response elements are often
referred to as transcription factors. Typically, transcription
factors contain at least two recognizable domains, a DNAbinding domain and an activation domain.
1. The DNA-binding domain binds to a specific nucleotide
sequence in the promoter or response element. Several types
of DNA-binding domain motifs have been characterized and
have been used to define certain families of transcription
factors. Some common DNA-binding domains include:
–
–
–
–
Zinc fingers (steroid hormone receptors)
Leucine zippers (cAMP-dependent transcription factor)
Helix-loop-helix
Helix-turn-helix (homeodomain proteins encoded by
homeotic/homeobox genes)
2. The activation domain allows the transcription
factor to:
a) Bind to other transcription factors
b) Interact with RNA polymerase II to stabilize the
formation of the initiation complex
c) Recruit chromatin-modifying proteins such as
histone acetylases or deacetylases
• Two types can be distinguished, general
transcription factors and specific transcription
factors. Examples are listed in Table 1-5-1.
Properties of Some Common Transcription Factors
Mouse Zif268
• Each “finger” binds small stretch of DNA
• Additive affect of each finger adds specificity and
binding affinity to entire domain
• Leucine zipper: Have basic amino acids with + charge
(bind DNA)
• E.g., cAMP dependent binding protein
• Helix-Loop-Helix: Have basic helix with + charge (bind
DNA)
• Best characterized in mammalian MyoD protein (involved
in muscle cell development)
• Helix-turn-helix DNA-binding
protein
• Extra N-terminal domain that
inserts into minor groove
• First eukaryotic examples found
fruit fly “homeotic” genes
– mutations in these genes cause
body-part changes
General Transcription Factors
• In eukaryotes, general transcription factors must
bind to the promoter to allow RNA polymerase II
to bind and form the initiation complex at the start
site for transcription.
• General transcription factors are common to most
genes. The general transcription factor TFIID (the
TATA factor) must bind to the TATA box before
RNA polymerase II can bind. Other examples
include SP-l and NF-l that modulate basal
transcription of many genes.
Specific Transcription Factors
• Specific transcription factors bind to enhancer regions
or, in a few cases, to silencers and modulate the
formation of the initiation complex, thus regulating the
rate of initiation of transcription.
• Each gene contains a variety of enhancer or silencer
sequences in its regulatory region. The exact combination
of specific transcription factors available (and active) in a
particular cell at a particular time determines which
genes will be transcribed at what rates.
• Because specific transcription factors are proteins, their
expression can be cell-type specific. Additionally,
hormones may regulate the activity of some specific
transcription factors. Examples include steroid receptors
and the CREB protein.
Peroxisome proliferator-activated receptors (PPARs) are
transcription factors that bind to DNA response elements
(PPREs) and control multiple aspects of lipid metabolism.
Individual members of this family of zinc-finger proteins are
activated by a variety of natural and xenobiotic ligands,
including:
–
–
–
–
Fatty acids
Prostaglandin derivatives
Fibrates
Thiazolidinediones
• The improvement in insulin resistance seen with
thiazolidinediones is thought to be mediated through their
interaction with PPARγ. Clofibrate binds PPARα affecting
different aspects of lipid metabolism than the
thiazolidinediones.
• An example of how response elements affect metabolism
can be seen in the pathway of gluconeogenesis (Figure
1-5-6).
• Gluconeogenesis is a hepatic pathway whose major
function is to maintain adequate glucose in the blood
for tissues like the nerves (brain) and red blood cells
during fasting. It also provides glucose during periods
of stress.
• Hormones that activate the pathway include:
– Glucagon secreted in response to hypoglycemia and
functioning via a membrane associated receptor that increases
cAMP concentration
 Cortisol secreted in response to stress, is permissive for glucagon in
hypoglycemia and acts through an intracellular receptor, which, like other
steroid receptors, is a zinc-finger DNA binding protein.
Control of Gluconeogenesis by Response Elements
Cortisol and Glucagon Stimulate Gluconeogenesis
Through Enhancer Mechanisms
• Phosphoenolpyruvate
carboxykinase
(PEPCK) catalyzes a critical reaction in
gluconeogenesis, which under many
conditions is the rate-limiting step in the
pathway.
• A cAMP response element (CRE) and a
glucocorticoid response element (GRE)
are each located upstream from the
transcription start site.
• Cortisol induces PEPCK gene expression by
the following sequence:
–
–
–
–
Cortisol diffuses into the hepatocyte
Binds to its receptor.
The complex enters the nucleus, and
Binds (through the zinc fingers) to the
glucocorticoid response element (GRE) associated
with the PEPCK gene, which
– Increases gene expression.
– PEPCK concentration increases in the cell.
– The rate of gluconeogenesis increases.
Glucagon induces PEPCK gene expression by the
following sequence:
• Glucagon binds to a receptor in the cell membrane.
• cAMP concentration increases.
• Protein kinase A becomes active, and then
• Phosphorylates and activates CREB.
• Activated CREB enters the nucleus and binds to the
CRE associated with the PEPCK gene, which
• Increases gene expression.
• PEPCK concentration increases in the cell.
• The rate of gluconeogenesis increases.
• These effects of CREB and the cortisol-receptor complex are not entirely
independent of each other. Each contributes, along with several other
transcription factors, to assembling a complex of activator proteins that
ultimately determine the level of PEPCK gene expression.
Control of Cell Differentiation by Homeodomain Proteins
During Development In Utero
• Sequential and coordinated gene expression is necessary for
proper tissue and cell differentiation during embryonic life.
• Groups of regulatory proteins called homeodomain proteins
are major factors in controlling this embryonic gene
expression.
• Each regulatory protein is responsible for activating a different
set of genes at the proper time in development.
• The regulatory proteins themselves are encoded by genes
called homeobox (HOX) or homeotic genes.
• Another closely related set of genes is the PAX (paired-box)
genes.
• Mutations in HOX or PAX genes might be expected to produce
developmental errors. Klein-Waardenburg syndrome (WS-III)
is one such developmental disorder resulting from a mutation
in a PAX gene.
Clinical Correlate
Klein-Waardenburg Syndrome
• All of the tissues affected in Klein-Waardenburg
syndrome are derived from embryonic tissue in
which PAX-3 is expressed. Symptoms include:
– Dystopia canthorum (lateral displacement of the inner
corner of the eye)
– Pigmentary abnormalities (frontal white blaze of hair,
patchy hypopigmentation of the skin, heterochromia
irides)
– Congenital deafness
– Limb abnormalities
Co-Expression of Genes
• Most eukaryotic cells are diploid, each chromosome
being present in two homologous copies. The alleles of
a gene on the two homologous chromosomes are
usually co-expressed.
• In a person heterozygous for the alleles of a particular
gene, for example a carrier of sickle cell trait, two
different versions of the protein will be present in
cells that express the gene.
• In the person heterozygous for the normal and sickle
alleles, about 50% of the β-globin chains will contain
glutamate and 50% valine at the variable position
(specified by codon 6).
Major exceptions to this rule of codominant
expression include genes:
• On the Barr body (inactivated X chromosome) in
women
• In the immunoglobulin heavy and light chain loci
(ensuring that one B cell makes only one
specificity of antibody)
• In the T-cell receptor loci
• Genomic Imprinting
Bridge to Medical Genetics
Genetic Imprinting in Prader-Willi Syndrome
• Genetic imprinting of a few gene regions results in mono-alleleic
expression.
• In Some cases this imprinting is according to the parent of origin.
The gene(s) involved in Prader-Willi syndrome is on chromosome 15
and is imprinted, so that it is normally expressed only from the
paternal, not the maternal, chromosome.
• In such a case, if one inherits a paternal chromosome in which this
region has been deleted, Prader-Willi syndrome results. It can also
result from uniparental (maternal) disomy of chromosome 15.
• Symptoms of Prader-Willi include:
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–
–
–
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Childhood obesity and hyperphagia
Hypogonadotrophic hypogonadism
Small hands and feet
Mental retardation
Hypotonia
Angelman Syndrome
• Characterized by unusual facial
appearance, short stature,
severe mental retardation,
spasticity, and seizures
• Have genetic info in 15q12
(12q11-q13) derived only from
father
Other Mechanisms
for Controlling
Gene Expression
in Eukaryotes
• Table 1-5-2
summarizes some
of the mechanisms
that control gene
expression in
eukaryotic cells.
Heme increases initiation of β–globin translation
• eIF2 is an initiation factor for protein synthesis. It transfers
bound initiator met tRNA to the 40S ribosomal subunitmRNA complex.
• During met-tRNA transfer, bound GTP is hydrolyzed to
GDP, which remains bound to eIF2.
• GTP must exchange with the bound GDP for the factor to
participate in another round of intiation. This exchange is
catalyzed by eIF2B, which is present in limiting quantities.
• eIF2 can be phosphorylated. In the phosphorylated form it
is very strongly bound by eIF2B. The binding strength is
high enough that the phosphorylated eIF2 is sequestered,
tying up much of the available eIF2B.
• In reticulocytes, the kinase that phosphorylates eIF2 is
HRI (heme-regulated eIF kinase). The kinase activity of
HRI is inhibited by binding of hemin.
• Hemin is an oxidation product of heme. It accumulates in
reticulocytes when heme production exceeds heme
utilization.
• Heme is utilized in the formation of hemoglobin by
binding stoichiometrically to globin polypeptides.
• This homeostatic mechanism is only possible in
cells such as reticulocytes that are geared to the
production of principally one protein.
Review Questions
Select the ONE best answer.
1. A culture of E. coli is grown in a medium
containing glucose and lactose. The
expression of the lactose operon over
time in the cells is shown in the graph
below. Which statement best describes
the change that occurred at point A?
A. Lactose was added to the culture
B. cAMP concentration increased in the cells
C. Glucose was added to the culture
D. Repressor protein dissociated from the operator
E. Repressor protein became bound to the operator
2. Klein-Waardenburg syndrome is a single-gene
disorder that includes dystopia canthorum (lateral
displacement of the inner corner of the eye),
impaired hearing, and pigmentary abnormalities.
The gene involved is most likely to be a
A. Pseudogene
B. Proto-oncogene
C. Transgene
D. Homeotic gene
E. Tumor suppressor gene
3. Escherichia coli cells grown in a medium with lactose as the
only carbon source are monitored for β-galactosidase activity
over time with the results shown below.
Which intracellular event would most likely be associated with
the change in enzyme activity observed?
A. Decreased synthesis of cytosolic cAMP
B. Activation of a repressor protein by lactose
C. Increased synthesis of a repressor protein
D. Dissociation of a cAMP-CAP complex from a CAP-binding
sequence
E. Binding of a repressor protein to an operator sequence