The document describes regulation of gene expression in prokaryotic and eukaryotic cells. In prokaryotes, gene expression is regulated through operons, clusters of genes that are coordinately controlled. The lac and trp operons in E. coli are discussed as examples, with the lac operon being inducible and the trp operon being repressible. In eukaryotes, gene expression can be regulated at many stages including chromatin modifications, transcription, RNA processing, translation and protein modification. This allows for cell specialization and differential gene expression with the same genome.

1. Lecture 26
Regulation of
gene expression
Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene
Gene available
for transcription
Transcription
RNA Exon
Primary transcript
Intron
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation Translation
of mRNA
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Active protein
Degradation
of protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)

2. Overview
• Prokaryotes and eukaryotes alter gene expression in
response to their changing environment
• In multicellular eukaryotes, gene expression regulates
development and is responsible for differences in cell
types
• RNA molecules play many roles in regulating gene
expression in eukaryotes

3. Regulation of gene expression
in prokaryotic cell - Operon units, system of
negative feedback
in eukaryotic cell come at any stage of gene
expression and proteosynthesis. Important are
noncoding RNAs.

4. Bacteria often respond to
environmental change by regulating
transcription
• Natural selection has favored bacteria that produce only
the products needed by that cell
• A cell can regulate the production of enzymes by
feedback inhibition or by gene regulation
• Gene expression in bacteria is controlled by the operon
model

5. Precursor
Feedback
inhibition
Enzyme 1
Enzyme 2
Enzyme 3
Tryptophan
Regulation of enzyme
activity
Regulation
of gene
expression
Regulation of enzyme
production
-
-
trpE gene
trpD gene
trpC gene
trpB gene
trpA gene

6. Operons: The Basic Concept
• A cluster of functionally related genes can be under
coordinated control by a single “on-off switch”
• The regulatory “switch” is a segment of DNA called an
operator usually positioned within the promoter
• An operon is the entire stretch of DNA that includes
the operator, the promoter, and the genes that they
control
Operon is a functional unit common in bacteria and phages. Activation
and inhibition of transcription are regulated in response of conditions
in environment.
Prokaryotic genetic information is not divided into introns and exons.

7. Operon
• is coordinately regulated clusters of genes,
which are transcribed into one mRNA
(polygenic mRNA)
• are genes for particular metabolic pathway
and are regulated by common promoter and
are ordered on DNA following each other

8. Escherichia coli
Lac operon, Trp operon – model systems =
metabolic pathways of
• utilization of lactose gen lacZ, lacY, lacA, catabolic
pathway with negative and positive regulation
• enzymes for TRP synthesis, anabolic pathway
with negative regulation

9. • The operon can be switched off by a protein repressor
• The repressor prevents gene transcription by binding to
the operator and blocking RNA polymerase
• The repressor can be in an active or inactive form,
depending on the presence of other molecules
• A corepressor is a molecule that cooperates with a
repressor protein to switch an operon off
• For example, E. coli can synthesize the amino acid
tryptophan

10. • By default the trp operon is on and the genes for
tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp repressor
protein, which turns the operon off
• The repressor is active only in the presence of its
corepressor--tryptophan; thus the trp operon is turned
off (repressed) if tryptophan levels are high

11. Promoter
DNA
Regulatory
gene
mRNA
trpR
5¢
3¢
RNA
polymerase
Protein Inactive
repressor
Promoter
trp operon
Genes of operon
Operator
Start codon Stop codon
mRNA 5¢
trpE trpD trpC trpB trpA
E D C B A
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor
(b) Tryptophan present, repressor active, operon off
No RNA
made

12. Repressible and Inducible Operons: Two
Types of Negative Gene Regulation
• A repressible operon is one that is usually on; binding
of a repressor to the operator shuts off transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off; a
molecule called an inducer inactivates the repressor
and turns on transcription

13. • The lac operon is an inducible operon and contains
genes that code for enzymes used in the hydrolysis and
metabolism of lactose
• By itself, the lac repressor is active and switches the lac
operon off
• A molecule called an inducer inactivates the repressor
to turn the lac operon on

14. Regulatory
gene
Promoter
Operator
DNA
lacI lacZ
mRNA
5¢
3¢
(a) Lactose absent, repressor active, operon off
lacI
RNA polymerase
(b) Lactose present, repressor inactive, operon on
No
RNA
made
RNA
polymerase
Active
Protein repressor
lac operon
DNA lacZ lacY lacA
mRNA
5¢
3¢
Protein
mRNA 5¢
Inactive
repressor
Allolactose
(inducer)
b-Galactosidase Permease Transacetylase

15. Lac operon - negative regulation
• regulatory gene produces repressor, which
binds operator and causes that RNAP is not
able to initialize transcription
• in the presence of lactose repressor is released
from operator. The repressor is changed by
inducer / lactose
RNA polymerase starts the transcription. In 2-3 minutes the amount of
enzymes is increased 1000x

16. • Inducible enzymes usually function in catabolic
pathways; their synthesis is induced by a chemical
signal
• Repressible enzymes usually function in anabolic
pathways; their synthesis is repressed by high levels of
the end product
• Regulation of the trp and lac operons involves negative
control of genes because operons are switched off by
the active form of the repressor

17. Positive Gene Regulation
• Some operons are also subject to positive control
through a stimulatory protein, such as catabolite
activator protein (CAP), an activator of transcription
• When glucose (a preferred food source of E. coli) is
scarce, CAP is activated by binding with cyclic AMP
(cAMP)
• Activated CAP attaches to the promoter of the lac
operon and increases the affinity of RNA polymerase,
thus accelerating transcription

18. Lac operon - positive regulation
• In the presence of glucose, E. coli preferentially uses
glucose for decomposing.
• If is low level of glucosis, the cAMP is increased.
• CAP (Catabolite activator protein) in the presence
of cAMP attaches promoter and activates the
transcription.
• CAP is allosteric regulatory protein
• When glucose levels increase, CAP detaches from the
lac operon, and transcription returns to a normal rate

19. Promoter
DNA
lacI lacZ
CAP-binding site
Operator
RNA
polymerase
binds and
transcribes
cAMP
Active
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
Promoter
DNA
lacI lacZ
CAP-binding site
Operator
RNA
polymerase less
likely to bind
Inactive lac
repressor
Inactive
CAP
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized

20. Summary
each operon consists of
• promoter (for RNA polymerase)
• operator (for repressor)
• several structural genes
• terminator
repressor = allosteric protein encoded by regulatory
gene
co-repressor = product molecule
inducer = substrate molecule

21. Eukaryotic gene expression is
regulated at many stages
• All organisms must regulate which genes are expressed
at any given time
• In multicellular organisms regulation of gene
expression is essential for cell specialization

22. Gene expression of eukaryotic cells
• each cell maintains specific program /
differential gene expression
• one mRNA carries information for one gene
(monogennic mRNA)
• posttranscription modifications of RNA
RNA processing and splicing
• regulation system is performed at the several
levels = transcription, translation, protein
activation + secretion

23. Differential Gene Expression
• Almost all the cells in an
organism are genetically
identical
• Differences between cell types
result from differential gene
expression, the expression of
different genes by cells with the
same genome
• Abnormalities in gene
expression can lead to diseases
including cancer
• Gene expression is regulated at
many stages
Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene
Gene available
for transcription
Transcription
RNA Exon
Primary transcript
Intron
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation Translation
of mRNA
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Active protein
Degradation
of protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)

24. Many steps at which eucaryotic gene
expression can be controlled

25. more complicated regulating system
• chromatin changes
• transcription
• processing RNA
• transport to cytoplasm
• degradation of mRNA
• translation
• cleavage, chemical modification
• protein degradation

26. 1. Chromatin changes
• Heterochromatin is highly condensed that is why
transcriptional enzymes can not reach the DNA
• Acetylation / deacetylation of histons
• Methylation [cytosin] - inactive DNA is highly
methylated
DNA methylation and histone de-acetylation repress
the transcription.

27. • DNA methylation
is esential for long-term inactivation of genes during
cell differentiation
Gene imprinting in mamals
• methylation constantly turns off the maternal or the
paternal allele of a gene in early development
• certain genes are expressed in a parent-of-origin-specific
manner
Epigenetic inheritance

28. Histone Modifications
• In histone acetylation, acetyl groups are attached to
positively charged lysines in histone tails
• This loosens chromatin structure, thereby promoting the
initiation of transcription
• The addition of methyl groups (methylation) can
condense chromatin; the addition of phosphate groups
(phosphorylation) next to a methylated amino acid can
loosen chromatin

29. Amino acids
available
for chemical
modification
Histone
tails
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription

30. DNA Methylation
• DNA methylation, the addition of methyl groups to
certain bases in DNA, is associated with reduced
transcription in some species
• DNA methylation can cause long-term inactivation of
genes in cellular differentiation
• In genomic imprinting, methylation regulates
expression of either the maternal or paternal alleles of
certain genes at the start of development

31. Epigenetic Inheritance
• Although the chromatin modifications just discussed do
not alter DNA sequence, they may be passed to future
generations of cells
• The inheritance of traits transmitted by mechanisms not
directly involving the nucleotide sequence is called
epigenetic inheritance

32. Regulation of Transcription Initiation
• Chromatin-modifying enzymes provide initial control
of gene expression by making a region of DNA either
more or less able to bind the transcription machinery

33. 2. Transcription
Transcription factors:
proteins that bind DNA and facilitate or inhibit RNA
polymerase to bind. They are a part of transcription initiation
complex.
general transcription factors for all protein-coding genes
specific transcription factors – transcription of particular
genes at appropriate time and place
- enhancers, activators, inhibitors, repressors

34. Organization of a Typical Eukaryotic Gene
• Associated with most eukaryotic genes are multiple
control elements, segments of noncoding DNA that
serve as binding sites for transcription factors that help
regulate transcription
• Control elements and the transcription factors they bind
are critical to the precise regulation of gene expression
in different cell types

35. Enhancer
(distal control
elements)
DNA
Proximal
control
elements
Transcription
start site
Upstream Promoter
Poly-A
signal
sequence
Exon Intron Exon Intron Exon
Transcription
termination
region
Poly-ADownstream
signal
Transcription
Exon Intron Exon Intron Exon
Cleaved
3¢ end of
primary
transcript
5¢
Primary RNA
transcript
(pre-mRNA)
Intron RNA
RNA processing
mRNA
Coding segment
G P P P AAA ××× AAA
5¢ Cap 5¢ UTR
Start
codon
Stop
codon 3¢ UTR
3¢
Poly-A
tail

36. The Roles of Transcription Factors
• To initiate transcription, eukaryotic RNA polymerase
requires the assistance of proteins called transcription
factors
• General transcription factors are essential for the
transcription of all protein-coding genes
• In eukaryotes, high levels of transcription of particular
genes depend on control elements interacting with
specific transcription factors

37. Enhancers and Specific Transcription Factors
• Proximal control elements are located close to the
promoter
• Distal control elements, groupings of which are called
enhancers, may be far away from a gene or even
located in an intron

38. • An activator is a protein that binds to an enhancer and
stimulates transcription of a gene
• Activators have two domains, one that binds DNA and
a second that activates transcription
• Bound activators facilitate a sequence of protein-protein
interactions that result in transcription of a given gene

39. • Some transcription factors function as repressors,
inhibiting expression of a particular gene by a variety of
methods
• Some activators and repressors act indirectly by
influencing chromatin structure to promote or silence
transcription

40. Activators
DNA
Enhancer Distal control
element
Promoter
Gene
TATA box
General
transcription
factors
DNA-bending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
RNA synthesis
Transcription
initiation complex

41. Cell-type specific transcription:
Genes encoding the enzymes of one metabolic
pathway are scattered over the different
chromosomes - coordinated control in
response of chemical signals from outside
the cell. The cell accept signals by receptors.
Signal transduction pathways activate
transcription activators or repressors.

42. Signal transduction pathways

43. Control
elements
Enhancer Promoter
Albumin gene
Crystallin
gene
LIVER CELL
NUCLEUS
Available
activators
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
LENS CELL
NUCLEUS
Available
activators
Albumin gene
not expressed
Crystallin gene
expressed
(b) Lens cell

44. Coordinately Controlled Genes in Eukaryotes
• Unlike the genes of a prokaryotic operon, each of the
co-expressed eukaryotic genes has a promoter and
control elements
• These genes can be scattered over different
chromosomes, but each has the same combination of
control elements
• Copies of the activators recognize specific control
elements and promote simultaneous transcription of the
genes

45. Nuclear Architecture and Gene Expression
• Loops of chromatin extend
from individual
chromosomes into specific
Chromosomes in the
sites in the nucleus
interphase nucleus
Chromosome
territory
• Loops from different
chromosomes may
congregate at particular
sites, some of which are
10 mm
rich in transcription factors
and RNA polymerases
• These may be areas
specialized for a common
Chromatin
Transcription
function
loop
factory

46. Mechanisms of Post-Transcriptional
Regulation
• Transcription alone does not account for gene
expression
• Regulatory mechanisms can operate at various stages
after transcription
• Such mechanisms allow a cell to fine-tune gene
expression rapidly in response to environmental
changes

47. 3. Processing RNA
• In alternative RNA splicing, different mRNA molecules are
produced from the same primary transcript, depending on which
RNA segments are treated as exons and which as introns
Exons
DNA
3
4
Troponin T gene
Primary
RNA
transcript
3
4
RNA splicing
1
1
2
2
mRNA or
3
2 2
1 1
4
5
5
5 5

48. 4, 5. transport of mRNA / degradation
• The life span of mRNA molecules in the cytoplasm is a
key to determining protein synthesis
• Eukaryotic mRNA is more long lived than prokaryotic
mRNA
• Nucleotide sequences that influence the lifespan of
mRNA in eukaryotes reside in the untranslated region
(UTR) at the 3¢ end of the molecule

49. 6. Translation
At the initiation stage – regulatory proteins bind the
5’ end of the mRNA with the cap.
Activation or inactivation of protein factors to initiate
translation

50. 7. Cleavage, chemical modifications
Cleavage
Post-translational modifications
Regulatory proteins [products] are activated
or inactivated by the reversible addition of
phosphate groups / phosphorylation
Sugars on surface of the cell / Glycosylation

51. • Polypeptide chain may
be cleaved into two or
three pieces
• Preproinsulin
• Proinsulin - disulfide
bridges
• Insulin
• Secretory protein

52. Post-translational modifications
Acid/base - act/inact
Hydrolysis – localization, act/inact
Acetylation - act/inact
Phosphorylation - act/inact
Prenylation - localization
Glycosylation - targeting

53. 8. protein degradation
• Lifespan of protein is strictly regulated
• Proteins are produced and degraded continually in
the cell.
• Proteins to be degraded are tagged with ubiquitin.
• Degradation of proteins marked with ubiquitin
occurs at the proteasome.

55. Chromatin modification
• Genes in highly compacted
chromatin are generally not
transcribed.
• Histone acetylation seems
to loosen chromatin structure,
enhancing transcription.
• DNA methylation generally
reduces transcription.
mRNA degradation
• Each mRNA has a
characteristic life span,
determined in part by
sequences in the 5¢ and
3¢ UTRs.
Transcription
• Regulation of transcription initiation:
DNA control elements in enhancers bind
specific transcription factors.
Bending of the DNA enables activators to
contact proteins at the promoter, initiating
transcription.
• Coordinate regulation:
Enhancer for
liver-specific genes
Enhancer for
lens-specific genes
RNA processing
• Alternative RNA splicing:
Primary RNA
transcript
mRNA or
Translation
• Initiation of translation can be controlled
via regulation of initiation factors.
Protein processing and degradation
• Protein processing and
degradation by proteasomes
are subject to regulation.
Chromatin modification
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation

56. Example of gene regulation

57. RReegguullaattiioonn && DDeevveellooppmmeenntt
• hhooxx GGeenneess
– CCoonnttrrooll OOrrggaann && TTiissssuuee DDeevveellooppmmeenntt
IInn TThhee EEmmbbrryyoo
–MMuuttaattiioonnss LLeeaadd TToo MMaajjoorr CChhaannggeess
• DDrroossoopphhiillaa WWiitthh LLeeggss IInn PPllaaccee ooff
AAnntteennnnaaee

58. RReegguullaattiioonn && DDeevveellooppmmeenntt

59. RReegguullaattiioonn && DDeevveellooppmmeenntt
hhooxx GGeenneess PPrreesseenntt IInn AAllll EEuukkaarryyootteess
– SShhoowwss CCoommmmoonn AAnncceessttrryy
–PPaaxx 66 hhooxx ggeennee
• CCoonnttrroollss eeyyee ggrroowwtthh iinn DDrroossoopphhiillaa,, MMiiccee
&& MMaann
• PPaaxx 66 ffrroomm MMoouussee PPllaacceedd IInn KKnneeee
DDeevveellooppmmeenntt SSeeqquueennccee OOff DDrroossoopphhiillaa
DDeevveellooppeedd IInnttoo EEyyee TTiissssuuee..
CCoommmmoonn AAnncceessttoorr >>660000MM YYeeaarrss AAggoo

61. Homeotic mutations transform
one body part into another.

62. Wild type Mutant
Eye
Antenna
Leg

63. The Regulation of Eukaryotic
Gene Expression
..using the example of PEPCK

64. PEPCK
• This is an acronym for an enzyme
• PhosphoEnol Pyruvate CarboxyKinase
• This enzyme is ONLY regulated by gene
expression!
• No allosteric activators, covalent
modification etc
• No activation by cAMP, inhibition by
insulin etc

65. PEPCK
• The enzyme is expressed in liver, kidney,
adipose tissue and to a lesser extent in
muscle
• It is a key enzyme in gluconeogenesis (the
synthesis of new glucose, usually from
lactate, pyruvate or alanine) and
glyceroneogenesis (the synthesis of
glycerol, usually from lactate, pyruvate or
alanine)

66. PEPCK overexpression in muscle
• a mouse with PEPCK overexpressed in muscle
only.
• This mouse was leaner than wild type mice, ran for
longer and lived longer!
• They were also more aggressive.
• The overexpression had switched the muscle fuel
usage to fatty acids with little lactate production.

67. The Supermouse….
• Eats 60% more food
than wild type mice
• Weighs 40% less than
wild type mice
• Can run for >4 h until
exhaustion whereas
the control littermates
stop after only 10 min
• Has 2 – 3 fold less
adipose tissue

68. PEPCK overexpression in adipose
tissue
• A mouse has the PEPCK enzyme
overexpressed in adipose tissue.
• The results couldn’t be further from
supermouse!

69. PEPCK overexpression in fat cells

70. PEPCK overexpression in adipose
tissue
• These mice are obese although
metabolically healthy (as measured by glucose
tolerance and insulin sensitivity) until you put them
on a high fat diet.
• Then you see insulin resistance and diabetes
emerging.

71. PEPCK overexpression in liver
• Leads to altered glucose tolerance
• Insulin resistance
• Increased gluconeogenesis causes increased
hepatic glucose production which is
released into the blood stream
• This caused increased insulin secretion but
ultimately insulin resistance.

72. PEPCK Knock out in liver
• Surprisingly these mice can maintain blood
glucose under starvation conditions
• They develop liver steatosis (fatty livers)
probably because of impaired oxidation of
fatty acids
• A total PEPCK knock out in all tissues is
lethal…mice die within days of birth.

73. Why the dramatically different
outcome for the mouse when PEPCK
is overexpressed in different tissues?
It is after all the same enzyme catalysing
the same reaction.

74. Glyceroneogenesis
COOH
C
CH2
PEPcarboxykinase
O
COOH
O C
H2C COOH
oxaloacetate
OAA
PO3
Phosphoenol pyruvate
PEP
COOH
C O
Pyruvate Carboxylase LDH
CH3
COOH
HC OH
CH3
NADH NAD+
CO2
Pyruvate Lactate
CO2
GDP
GTP
Alanine

75. Glyceroneogenesis
Fatty acids H3C CO
CH2OPO3
C O
CH2OH
CH2OPO3
HC OH
C
O
Glyceraldehyde
3-P
Dihydroxyacetone
phosphate (DHAP)
H
CH2OPO3
HC OH
CH2OH
Glycerol 3-P
Triglycerides
S-CoA
PEP