Regulation of Gene Expression

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 Case report
• A 10 year old boy presented with signs and
symptoms and subsequent laboratory
investigation reports suggestive of Acute
lymphoblastic leukaemia. Patient was
receiving Methotrexate as a part of this
treatment. After several weeks of
chemotherapy with methotrexate, the boy
showed signs of resistance to treatment. What
could be the most likely mechanisms to
explain this resistance to methotrexate?
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 Objective
• What is Gene expression
• Necessity of regulation of gene expression
• Different types of gene regulation
• Gene regulation in prokaryotes
– Lac operon
– Tryptophan operon
• Gene regulation in Eukaryotes
– At transcription level
– At translation
– Modification of DNA

• DNA regulatory proteins

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• What is gene expression?
– Formation of gene products
• RNA and Protein
• Necessity of Gene Expression and regulation
– Normal growth , development and functioning of
an organism
– Production of gene product according to the
requirement of the body

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 Type of gene expression

• Constitutive
– Occur all the time and at a relatively constant rate

• Regulated
– Transcribed only when needed

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 Regulation of gene expression

• Positive regulation---- Enhancer/Activator

• Negative regulation----Repressor/ Silencer

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 Temporal responses to regulatory signal

Type A

observed in prokaryotes in response to sudden changes of

the intracellular concentration of a nutrient
in higher organisms after exposure to inducers such as
hormones, nutrients, or growth factors 7
 Type B

occurs during development of an organism, when only the

transient appearance of a specific gene product is required

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 TypeC

Occurs during the development of differentiated function

in a tissue or organ
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 Stages of regulation of gene
expression
• 1. Transcription
• 2. Post transcriptional modification
• 3. Translation
• 4. Post translational modification
• 5. RNA degradation
• 6. Protein degradation

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 Few specialized genetic and regulatory terms
Operon
groups of genes sequentially arranged
on the chromosome along with the regulatory
elements that determine their transcription

Polycistronic mRNA

Constitutive /House keeping gene

Expressed at a constant level

Inducible gene/Regulated egne

expressed only under certain condition
Gratuitous inducer: Acts as inducer but not as substrate
An example is isopropylthiogalactoside (IPTG)
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Cis acting
elements and
Trans acting
molecules

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 Regulation of Prokaryotic Gene
Expression
• Transcription is the primary site of regulation.

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Role of Promoters

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 Prokaryotic promoters share two regions of highly
conserved nucleotide sequence

Promoter: A regulatory region o DNA that serves to bind RNA polymerase

II that in turn binds other substances that will lead to initiation of transcription

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Adapted from Harper’s Biochemistry
 Operon Model

Described by Jacob and Monod in 1961

Hypothesis: Based on the regulation of lactose metabolism by the

intestinal bacterium E coli

Led to the discovery of basic principles of Gene

transcription activation and Repression

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 Type of Operon
• Catabolic Operon
– Regulates the gene expression whose product have a
catabolic role
– e,g. Lac operon

• Anabolic Operon
– Genes of anabolic pathway such as synthesis of amino
acids are coordinately regulated
– e,g. Tryptophan operon
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Lac Operon

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LacI gene encoding repressor
CRE: cAMP response element
Regulatory region : Promoter : RNAP binds
Operator: Repressor binds
Structural genes:

lac Z: β Galactosidase
lac Y: Permease
lac A:Thiogalactoside transacetylase
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• The operator locus is a region of double-stranded DNA that
exhibits a twofold rotational symmetry and an inverted
palindrome (indicated by arrows about the
dotted axis) in a region that is 21 bp long, as shown below:

The binding occurs mostly in the major groove

The operator locus is between the promoter site and the
transcription initiation site of the lacZ gene

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Lactose acts both as inducer and substrate
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 Tryptophan Operon
• Trp operon contains 5 structural genes
• Trp operon is subject to negative control
• Trp facilitates the binding of the repressor to the
operator
• Trp is a corepressor
• Trp operon also regulated by attenuation
– Transcription initiated but terminated well before
completion
Transcriptional attenuation occur in Prokaryotes
but not in Eukaryotes

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Attenuation of transcription of the trp
operon when tryptophan is plentiful.

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 Coordination of transcription and
translation in prokaryotes
• 1. Stringent response

•
. 2. Regulatory ribosomal proteins

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Regulation of
transcription by the
stringent response
to amino acid
starvation

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Regulation of translation by an
excess of ribosomal proteins.

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 MCQ
• Which of the following mutations is most
likely to result in reduced expression of the lac
operon?
• A. i– (no repressor protein made)
• B. Oc (operator cannot bind repressor protein)
• C. Cya– (no adenylyl cyclase made)
• D. Functionally impaired glucose transporter

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 Regulation of Eukaryotic Gene
Expression
• Transcription is the primary site
of regulation

• Role of Promoter

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Schematic showing the transcription control regions in
a hypothetical mRNA-producing eukaryotic gene
transcribed by RNA polymerase II

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The eukaryotic basal transcription complex

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 Stages of Regulation of Eukaryotic
Gene Expression
• A. Coordinate regulation
– 1. Galactose circuit
• Coordinated expressin mediated by Gal4
– 2.Hormone response system
• Intracellular receptors
• Cell surface receptors

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Regulation of Galactose circuit in yeast

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Transcriptional regulation by intracellular steroid hormone
receptors.
GRE = glucocorticoid-response element
(an example of a hormone response element); GR = glucocorticoid
receptor. 35
Transcriptional regulation by
receptors located in the cell
membrane.

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 Regulation of Eukaryotic Gene
Expression

• B. Regulation by co- and posttranscriptional

processing of mRNA
– 1. Splice-site choice/ Alternate splicing
• Tissue-specific protein products
• tropomyosin (TM) regulates the functions of actin
in both muscle and nonmuscle cells

– 2. Alternative polyadenylation
– 3. mRNA editing

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RNA editing of apo B in the intestine and generation of the
apo B-48 protein needed for chylomicron synthesis
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 – 4. mRNA stability
• A. IRON Metabolism
– Regulation by mRNA degradation—Regulation of transferrin
receptor (TfR) synthesis

IRE = iron-responsive element; IRP = iron-responsive element binding protein. 39

• Ferritin synthesis is regulated at the level of
translation
– Ferritin mRNA contains IRE at 5’ UTR
– Binding with IRP prevents translation because it overlaps the
translation initiation site
– Iron availability is more: Fe binds to IRP-allows translation of
free mRNA of ferritin

– Iron metabolism involves both mRNA stability and translation

level

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 3. mRNA stability

--b. RNA interference (RNAi) by

cleavage of target mRNA.

miRNA
si RNA

RNAi therapy:
Age-related macular degeneration (AMD) is triggered
by overproduction of vascular endothelial growth factor
(VEGF). The siRNA drug targets the mRNA of VEGF and
Promotes its degradation- injected in eyes.

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C. Regulation at Translation level

Regulation of translation initiation in eukaryotes by phosphorylation

of eIF-2. RER = rough endoplasmic reticulum.
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 D. Regulation through modifications
to DNA
• 1. Access to DNA
• 2. Amount of DNA:
• 3. Arrangement of DNA
• 4. Mobile DNA elements

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 1. Access to DNA
• Histone acetylation/deacetylation by the
histone acetyltransferase and histone
deacetylase enzymes

• Extent of methylation of cytosine bases in CG

rich regions (CpG islands) in the promoter
region by methyltransferases that use S-
adenosyl - methionine as the methyl donor

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 2. Amount of DNA:
• Gene amplification : increase in copy
Number seen in response to particular
chemotherapeutic drugs such as methotrexate
 inhibitor of the enzyme dihydrofolate
reductase (DHFR),
 required for the synthesis of thymidine
triphosphate (TTP)
 in the pyrimidine biosynthetic pathway

• Resistance to the drug

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 3. Arrangement of DNA

V=Variable, D= Diversity, J= Joining

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 4. Mobile DNA elements
• Transposons comprise 50% 0f human genome
• Movement is mediated by transposase
• Movement can be
– Direct
– Replicative
• Retrotransposon (90% of transposon)
• Hemophilia, Duchenne Muscular dystrophy
• Antibiotic resistance –Exchange of Plasmids containing
Tn carrying antibiotic resistance genes

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 Summary

• Gene expression results in the production of a functional gene product

(either RNA or protein
• Genes can be either constitutive or regulated
• Regulation of gene expression occurs primarily at the level of
transcription in both prokaryotes and eukaryotes
• mediated through the binding of trans-acting proteins to cis-acting
regulatory elements on the DNA
• In eukaryotes, regulation also occurs through modifications to the DNA,
as well as through posttranscriptional and posttranslational events
• In prokaryotes regulation is achieved through operons
• Operon: groups of genes sequentially arranged on the chromosome along
with the regulatory elements that determine their transcription
• operon is induced by an isomer of lactose (allolactose)
• The trp operon is also regulated by attenuation

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• Transcription of rRNA and tRNA is selectively inhibited in prokaryotes by
the stringent response to amino acid starvation
• Gene regulation is more complex in eukaryotes. Operons are not present,
but coordinate regulation of the transcription achieved through the
binding of trans-acting proteins to cis-acting elements
• In multicellular organisms coordinated regulation, achieved through
binding of hormone receptor–hormone complex to the DNA
• Co and posttranscriptional regulation is also seen in eukaryotes
• Regulation at the translational level can be caused by the
phosphorylation (inhibition) of eIF-2.
• Gene expression in eukaryotes is also influenced by availability of DNA to
the transcriptional apparatus

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 MCQ1
• Which of the following is the basis for the
intestinespecific expression of apoprotein B-
48?
• A. DNA rearrangement and loss
• B. DNA transposition
• C. RNA alternative splicing
• D. RNA editing
• E. RNA interference
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 MCQ 2
• Which of the following is best described as
being trans-acting?
• A. CAP site
• B. Operator
• C. Promoter
• D. Repressor

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 MCQ 3
• Which of the following is most likely to be true in
hemochromatosis, a disease of iron accumulation in the
body?
• A. The mRNA for the transferrin receptor (TfR) is stabilized
by the binding of IRPs to 3' stem-loop structures known as
IREs.
• B. The mRNA for the TfR is not bound by IRPs, and is rapidly
degraded.
• C. The mRNA for apoferritin is not bound by IRPs at its 5'
stem-loop IRE, and is translated.
• D. The mRNA for apoferritin is bound by IRPs, and is not
translated.
• E. Both B and C

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 MCQ 4
• After several weeks of chemotherapy with
methotrexate, a cancer patient’s tumor begins to show
signs of resistance to treatment. Which of the following
mechanisms is most likely to explain this resistance to
methotrexate?
• A. Deficiency of thymidylate synthase
• B. Overproduction of xanthine oxidase.
• C. Deficiency of PRPP synthase.
• D. Deficiency of thymidine kinase.
• E. Overproduction of dihydrofolate reductase

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 DNA regulatory protein
• Proteins that regulate gene expression:
Repressor, enhancer, silencer
• These proteins show several motifs which are
involved in these DNA protein interaction
• Only a small part of the protein has direct
contact with DNA
• Interactions are reversible and involve non
covalent bonds
• Some commonly seen structural motifs
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• Helix turn helix motif
• Zinc finger motif
• Leucine Zipper motif

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Helix turn helix motif

E,g. Lambda repressor, CAP protein, Tryptophan repressor, Homeobox protein in mammals
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Zinc fingers are a series of repeated domains (two to nine) in which
each is centered on a tetrahedral coordination with zinc.

Steroid thyroid receptor protein and calcitriol (vitamin D) receptor protein

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The leucine zipper motif

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A schematic model of the DNA-binding domain of C/EBP

cfos.cjun or cjun.cjun 59
Transcription Factors That Contain DNA Binding Motifs

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 References
• Harper’s Illustrated Biochemistry-30th edition
• Lippincott’s Illustrated Reviews: 5th edition
• Lehninger Principles of Biochemistry
• Biochemistry 4th edition Donald Voet and
Judith G. Voet.

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