15 Regulation of Gene
Expression
Expression of genetic information is dependent
on regulatory mechanisms that either activate
or repress the transcription of genes.
Transcription is modulated by the
interaction at various regulatory molecules
with DNA sequences, most often located
upstream from affected genes.
Genetic regulation in eukaryotes also occurs
during post transcriptional events.
•Translation
Gene expression
–Initiation
• Transcription
–Elongation
– Initiation Elongation Termination
Jacques Monod
–Termination
Francois Jacob
• 15.1 Genetic Regulation in Prokaryotes:
An Overview
15.2 Lactose Metabolism in E. coli: An
Inducible System
Structural Genes
The Discovery of Regulatory Mutations
The Operon Model: Negative'Control
Genetic Proof of the Qperon Model
Isolation of the lac Represser
The CAP Protein: Positive Control of the lac Operon
• 15.3 Tryptophan Metabolism in E. coli:
A Repressible Gene System
Genetic Evidence for the trp Operon ,Attenuation
15.4 Genetic Regulation in Eukaryotes:
An Overview
• 15.5 Regulatory Elements, Transcription
Factors, and Eukaryotic Genes
Promoters
Enhancers
Transcription Factors
Structural Motifs of Transcription Factors
Assembly of the Transcription Complex
Chromatin Conformation, DNA Methylation,
and Gene Expression
15.6 Gene Regulation by Steroid Hormones
15.7 Posttranscriptional Regulation of Gene
Expression:
Alternative Splicing of mRNA
15.1 Genetic Regulation in Prokaryotes:
An Overview
• Regulation of gene expression has been studied
extensively in prokaryotes, particularly in
Escherichia coll. Highly efficient mechanisms
have evolved that turn genes on and off,
depending on the cell's metabolic needs in
particular environments. Detailed analysis of
proteins in E. coll has shown that for the more
than 4000 polypeptide chains encoded by the
genome, a vast range of concentration of gene
products exists. Some proteins may be present in
as few as 5-10 molecules per cell, whereas others,
such as ribosomal proteins and the many proteins
Regulation of Gene Expression
• At Transcription stage:
– Economical, but can’t be reversed quickly
• At Translation stage:
– Wasteful, but easily reversible
Regulation of Gene Expression
Feedback regulation:
– Positive control: the gene is off unless it is
turned on
– Negative control: the gene is on unless it is
turned off
Regulation of Gene Expression
Feedback regulation:
– Induction: the “target” molecule turns
expression ON (e.g., by disactivating the
repressor)
– Repression: the “target” molecule turns
expression OFF
The lac Operon in E. coli
Genetic System under both
Induction and Repression regulation.
Let’s first consider Induction.
Cis- and trans- acting regulatory
sequences
• Operator: a cis-acting element
Influences expression of genes downstream
from it on the same 2-stranded DNA
• Repressor gene: a trans- acting regulatory gene
Influences expression of any relevant genes in
the same cell, on the same or different DNA
Let’s stop here and solve some
problems
• You have 2 constitutive lac mutants (always
expressing lac operon). You transform them with a
plasmid containing an intact lac operon. One of
them changes the phenotype to wild type. The
other does not no matter how much you try. What
may be the genotypes of these 2 mutants?
Let’s stop here and solve some
problems
• You have a mutant strain with a mutation in lac
operon repressor gene, which prevents lactose
from binding to repressor protein.
• What will be the phenotype? Can it be changed by
transforming this strain with a plasmid containing
any lac operon structural or regulatory genes?
Homework!
• Textbook, Ch. 17, p.459: #5, 6, 10(extra
spicy, optional)
• Problem manual, p.82-83: #44, 48 (solved)
Pop-up quiz:
Genotype
I- P+ O+ Z+
(I- codes for inactive
repressor protein)
I- P+ O+ Z+/
I+ P+ O+ Z-
Phenotype
Phenotype
(lactose present) (lactose absent)
CAP-cAMP control
Glucose present ->
cAMP level decreases ->
no CAP-cAMP complex is formed ->
no CAP binding to promoter ->
no activation of transcription
Let’s compare two controls of lac
operon
• Repressor-operator control
• CAP-cAMP control
Let’s compare two controls of lac
operon
• Repressor-operator control
Negative control
Control molecule: lactose
• CAP-cAMP control
Positive control
Control molecule: glucose
Another operon under double
control:
Trp operon
Trp = triptophan
Let’s compare regulation of of
lac operon and trp operons
• lac operon
Induction (target molecule, lactose,
induces expression by desactivating
repressor protein)
• trp operon
Repression (target molecule, triptophan,
represses expression by activating repressor
protein)
But.. It’s not the whole story!
Attenuation regulation of trp-operon
Regulation of Gene Expression
in Prokaryotes
Hartwell pp. 551-560, 567-571
Regulation of transcription
• Differences in the basepairs in the -35 and -10
boxes allow genes to be expressed at different
levels.
• This kind of regulation is important, but it does
not allow the cell to adjust its pattern of gene
expression in a dynamic manner to meet
changing needs and environmental stresses.
Strategies for regulating gene
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Strategies for regulating gene
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Switches in the s subunit of RNA polymerase
• Under certain conditions, the cell needs to
induce a large set of genes that are normally
silent
• Examples include:
– heat-shock proteins
– nitrogen starvation
– developmental changes such as sporulation
Switches in the s subunit of RNA polymerase
• Under certain conditions, the cell needs to
induce a large set of genes that are normally
silent
• This can be accomplished by synthesizing
or activating a different RNA polymerase s
subunit that recognizes a distinct set of
promoter sequences.
s32 -
s70 s54-
pg. 786
Strategies for regulating
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
In prokaryotes, genes with related
functions can be expressed as a single
mRNA controlled by one
promoter
Promoter
Transcription
P
O
LacZ
b-galactosidase
LacY
lactose permease
The lac operon
LacA
unknown function
Operons are regulated by two
kinds of elements
RNAP
Transcriptional regulatory proteins
Transcription
Promoter
Operator
Regulatory sites
Gene 1
Transcriptional regulatory proteins
• Often called transcription factors
• Some transcriptional regulatory proteins
stimulate transcription initiation (called
activators)
• Other transcriptional regulatory factors inhibit
transcription (called repressors)
• In bacteria, the sites where transcription
factors bind reside close to the transcription
start site. These binding sequences are called
operators.
The regulatory gene for an operon
can reside at another site of the
chromosome
pg. 869
The Lac Operon:
A classic example of dynamic regulation of
gene expression
• b-galactosidase
pg. 868
The addition of lactose to E. coli leads to a
large increase in b-galactosidase synthesis
b-gal synthesis stops
b-gal synthesis is approximately
6% of total protein synthesis
(cell growth)
- b-galactosidase expression is inducible.
pg. 869
The addition of lactose to E. coli leads to a
large increase in b-galactosidase synthesis
b-gal synthesis stops
b-gal synthesis is approximately
6% of total protein synthesis
(cell growth)
-Note: The increase in b-galactosidase synthesis only occurs in the
absence of preferred carbon/energy sources such as glucose.
pg. 869
Isolation of mutations affecting
the induction of the lac operon
Inducers of the lac operon
-the natural inducer of lac operon expression
-formed from lactose by an activity of
b-galactosidase
1,6-Allolactose
-a synthetic inducer of lac operon expression
-cannot be metabolized
IPTG
Isolation of mutations affecting
the induction of the lac operon
Assaying for b-galactosidase activity using X-Gal substrate
Isolation of mutations affecting
the induction of the lac operon
Assaying for b-galactosidase activity using X-Gal substrate
wild-type E. coli grown on media
with X-gal but without glucose
wild-type E. coli grown on media
with X-gal and IPTG but without glucose
Isolation of mutations affecting
the induction of the lac operon
Constitutive mutant
a mutant that expresses
b-galactosidase without inducer
mutagenize
plate on media
with X-gal and
lacking IPTG and
glucose
Isolation of mutations affecting the
induction of the lac operon
Uninducible mutant
a mutant that fails to express
functional b-galactosidase
mutagenize
plate on media
with IPTG, X-gal
and lacking glucose
• Most of these mutation have
inactivated the lacZ gene rather
than affected its expression.
• However, there are mutants in
which the expression of lacY
and lacA have also been lost.
Production of B-galactosidase by strains
grown under different conditions
IPTG
-
-
+
+
Glucose
-
+
-
+
WildType
no
no
yes
no
LacZ-
no
no
no
no
oc
yes
no
yes
no
i-
yes
no
yes
no
is
no
no
no
no
Strain:
Genetic analysis of lac operon regulation
identifies two key components
• Transcriptional regulatory protein
– encoded by the lacI gene (maps near the lac operon)
– loss-of-function lacI (i-) mutations cause the lac operon
to be constitutively expressed
• This indicates that LacI acts as a repressor of lac operon
transcription in the absence of inducer
• Site for lac repressor binding
– called the lac operator (lacO)
– mutations of the lac operator (oc) also lead to failure of
lac repressor binding and constitutive expression
Regulation of the lac operon
pg. 872
Using bacterial merodiploids to perform
complementation tests
• E. coli are haploid,
lac
lac
F factor
E. coli chromosome
• but some E. coli have large plasmids called F factors that can be
transmitted (mated) from one E. coli to another.
• Through complicated genetic tricks, it is possible to isolate
strains in which the F factor contains a large region of E. coli
chromosome including the lac operon.
• Such a partially diploid strain is called merodiploid.
Cis vs. Trans
Transcription
Promoter Operator
Allele 1
Transcriptional regulatory protein
Transcription
Promoter Operator
Allele 2
• Trans-acting: In a cell with two copies of an operon, the
transcriptional regulatory protein will regulate the
expression of either allele,
• Cis-acting: An operator (the site where a transcriptional
regulatory protein binds) can only affect the allele to which
it is joined.
Evidence that lacI encodes a repressor:
the LacI protein can act in trans
lacI-, lacO+, lacZ+
lacI+, lacO+, lacZ-
F factor
Constitutive
Uninducible
• Result: Inducible synthesis
Evidence that lacO acts in cis
lacI+, lacO-, lacZ-
lacI+, lacO+, lacZ+
F factor
inducible
lacI+, lacOc, lacZ+ lacI+, lacO+, lacZ-
F factor
constitutive
• Dominant mutations that gave uninducible lac operon
expression were also isolated in the lacI gene.
• These encode a “super repressor” (is) that cannot bind the
inducer and therefore is always bound to the operator
Biochemical analysis of
LacI repressor action
• Identification of the lac operator sequence
Biochemical confirmation that LacI repressor acts by
binding to the lac operator in the absence of the inducer
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Hartwell, pg. 540
Lac repressor binds the operator
as a dimer
The lac repressor binding site
overlaps the RNA polymerase and
transcription initiation sites
Hartwell, pg. 543
Summary of analysis of LacI
repressor action
• Results
– Lac repressor binds the operator region.
– Constitutive mutants
• change the operator so that it cannot bind repressor (oc)
• change the repressor so that it cannot bind the operator (i-)
– The addition of inducer prevents lac repressor from
binding the operator.
– Uninducible mutants (is) change the repressor so that
it cannot bind inducer (and therefore stays bound to
the operator)
Strategies for regulating
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Catabolite Activator Protein
Transcription
LacI
P
O
LacZ
LacY
LacA
• Even in the presence of lactose, the lac operon is
poorly expressed if a preferred carbon/energy
source such as glucose is present.
• The lac operon promoter is very weak and requires
another transcriptional regulatory protein to be
strongly expressed.
• This protein is called catabolite activator protein
(CAP).
Catabolite activator protein
• E. coli has a system for monitoring its energy
levels.
• When energy stores are low, the enzyme
adenylcyclase catalyzes the synthesis of cyclic
AMP from ATP.
• cAMP serves as a signal to turn on operons
responsible for digesting (catabolizing) lesspreferred sugars such as lactose.
Catabolite activator protein
cAMP
cAMP
CAP
CAP
Transcriptional activator
Catabolite activator protein
• CAP-cAMP binds to the promoter next to the
RNA polymerase binding site.
• CAP makes contacts with RNA polymerase that
increase the binding of RNA polymerase to the
lac promoter.
Hartwell, pg. 543
lactose and glucose
Off
glucose without lactose
Off
no lactose or glucose
Off
lactose without glucose
On
Strategies for regulating gene
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
The trp operon:
a classic model for attenuation
The trp operon
• The trp operon encodes the enzymes required
to synthesize tryptophan (TrpA-E).
• Synthesis of the trp mRNA is controlled by a
repressor that blocks transcription when bound
to tryptophan.
• Therefore, when tryptophan levels are high,
transcription will be repressed.
Hartwell, p. 546
The Trp operon is regulated by a repressor
Hartwell, p. 546
• However, in the absence of trp repressor, the
synthesis of trp mRNA is still partially
repressed by the presence of tryptophan in the
growth medium.
Hartwell, p. 546
Attentuation: The trp operon mRNA contains a leader
region that can fold into different stem-loop structures
Trp codons in the leader sequence sense
the availability of tryptophan
• The trp mRNA leader contains two trp codons
in a row.
• When the tryptophan supply in the cell is
limited, ribosomes will stall at these trp codons.
• The ability of the ribosome to read through
these codons regulates the stem-loop choice.
The availability of tryptophan regulates
which stem loop structure forms
Other operons encoding amino acid
biosynthesis enzymes are also
regulated by attentuation
p. 888
Strategies for regulating gene
expression in prokaryotes
• Switches in the s subunit of RNA polymerase
• Control by a regulated repressor of transcription
• Control by a regulated activator of transcription
• Regulated attenuation (termination) of transcripts
Regulation of Gene Expression
in Eukaryotes
Hartwell pp. 250-256, 581-590, 602-609
Transcription and translation
in prokaryotes vs eukaryotes
• In prokaryotes, transcription and translation are
tightly coupled.
• In contrast, transcription and translation are
spatially separated in eukaryotes.
Primary RNA transcripts are extensively
modified before leaving the nucleus
• Nascent mRNA is “capped” with a 7methylguanosine at the 5’ end
• 3’ end is polyadenylated (100-200 residues) to
protect against degradation
• mRNA editing can occur in rare cases
• Splicing: often introns must be removed from
transcript and protein-coding exons spliced
together to form mature mRNA for translation
Primary RNA transcripts are extensively
modified before leaving the nucleus
• 5’ capping
–
–
–
–
protects mRNA from ribonucleases
promotes translation
Capping enzyme adds backward G
Then methyl transferases modify G
and 1 or more nucleotides
First base of transcript
Primary RNA transcripts are extensively
modified before leaving the nucleus
• 3’ polyadenylation
– Cleavage and polyA
addition are initiated via
recognition of AAUAAA
site 11-30 nucleotides
upstream of tail
– Protects RNA from rapid
degradation
– May aid transport out of
nucleus
– May increase efficiency of
translation
Primary RNA transcripts are extensively
modified before leaving the nucleus
• mRNA editing
– only a few examples are known
synthesized in the liver
deaminase is only
present in small intestine
synthesized in the intestine
Primary RNA transcripts are extensively
modified before leaving the nucleus
• mRNA splicing
• Some parts of the RNA
transcript are removed
prior to translation
• the excised pieces are
called introns
(intervening)
• the remaining pieces
are called exons
(expressed)
The mechanism of RNA splicing
• Certain sequences are conserved near splicing
junctions and serve as splicing signals
• Some key bases are in the intron and others are in
the flanking exons
The mechanism of splicing
• Splicing involves the excision of the intron in a
lariat form.
The mechanism of splicing
• Splicesomes are large RNA- and protein-containing
complexes.
• Some of the RNAs are used to recognize the splice
junctions.
• Example: U1 snRNA recognizes 5’ splice site
The mechanism of splicing
• Splicesomes are large RNA- and protein-containing
complexes
• Some of the RNAs are used to recognize the splice
junctions
• Example: U2 snRNA recognizes branch site
Spliceosome Assembly
Why splicing?
• mRNA splicing can allow a single gene to express
several different proteins (isoforms)
• These different forms often have different activities,
can interact specifically with different molecules
– For example, splicing can result in secreted vs membrane
bound forms of a protein like an antibody
• The production of different isoforms can be regulated
during development
– The Drosophila sex determination system provides several
classic examples of developmentally regulated alternative
splicing giving rise to differing functions
Splicing can produce a secreted
vs membrane-bound antibody
• Alternative splicing of this
heavy chain gene can
produce two different
types of antibody with the
same specificity
• When activated by antigen,
B-cells begin secreting
their antibodies
• Secreted form lacks exons
7 and 8 which encode
membrane attachment
domain
Drosophila Doublesex is alternatively
spliced into male or female forms
Functional Transformer
activates female-specific genes
and represses male-specific genes
Doublesex female isoform
Translation start
1
Stop
Stop
2
3
4
5
6
Doublesex male isoform
No functional Transformer
activates male-specific genes
and represses female-specific genes
Regulation of transcriptional
initiation in eukaryotes
• Three different RNA polymerases transcribe
genes in eukaryotes
– RNA polymerase I transcribes rRNAs (18S,
5.8S and 28S)
– RNA polymerase II transcribes mRNAs and
snRNAs
– RNA polymerase III transcribes tRNAs and 5s
rRNAs
• Each polymerase consists of many subunits.
Promoter elements in prokaryotes
• In prokaryotes, the promoter elements are welldefined.
• They are always present and always in the same
position relative to the start site of transcription.
Promoter elements in eukaryotes
• In eukaryotes, the promoter elements are much
less well-defined.
frequently not present
usually present
• They are not always present and can be various
distances from the start site of transcription.
Transcription factors TFIIA, B, D, E
and F play important roles in transcript
initiation by RNA polymerase II
• These factors were identified by purifying the
proteins required for correct initiation in vitro.
• Each factor is composed of multiple proteins.
• TFIID contains the 30 kilodalton (kD) TATA-box
binding protein.
• This complex of proteins together is called the
basal transcriptional machinery.
Sequential
assembly
of the
basal
transcription
machinery
Enhancer elements control the
activity of eukaryotic promoters
• Eukaryotic promoters are invariably weak and require
“enhancer elements” to drive high level expression.
• Enhancer elements are defined as DNA sequences that
promote the expression of a gene.
– Enhancer elements are distinguished from promoters in that
their function does not depend on their orientation relative to
the transcription units.
Enhancer
(TATA)
or
(TATA)
Enhancer
Enhancer elements control the
activity of eukaryotic promoters
• Enhancer elements do not need to be placed at a precise
distance from the promoter.
Enhancer
(TATA)
or
Enhancer
(TATA)
• Enhancer elements are frequently tens of kilobasepairs
(Kb) away from the transcriptional start site.
Enhancer elements contain multiple binding
sites for transcriptional activator proteins
• Many developmentally important genes have
enhancers with binding sites for many different
transcriptional activator proteins.
• Transcriptional activator proteins frequently
consist of two modular domains:
• Many transcriptional activators have negativelycharged regions that may interact with
components of the basal transcription machinery.
• Removing the activation domain can convert a
transcriptional activator into a repressor
DNA looping allows transcriptional activator
proteins to interact with the TFII components
• Silencer elements that provide binding sites for
transcriptional repressor proteins also exist
Repressor proteins reduce transcription
through competition or quenching
• Repressor can
compete with activator
for enhancer site
• Repressor can quench
activator by
– Blocking DNA-binding
domain
– Blocking activation
domain
The expression pattern of an individual gene
is determined by the transcription factors
that bind its enhancers and their regulation
How is the activity of enhancers
regulated during development and in
response to environmental changes?
• Many types of regulation are observed:
– tissue-specific expression
• Many transcriptional activators are only expressed in specific cell types.
– by binding to hormones
• For example, steroid receptors are enhancer binding proteins whose activity
depends on hormone binding.
– by phosphorylation
• Many transcription factors are only active when phosphorylated. These
include transcription factors that respond to hormones and growth factors
such as insulin.
• Together, these kinds of regulation can limit
gene expression to specific times and tissues.
The GAL4, GAL80
Gene System
• One enhancer controls the
genes GAL1,7, and 10
• Its activator, GAL4, is
quenched by GAL80 in
absence of galactose
• GAL1 and GAL3 can be
induced by galactose to bind
GAL80 and prevent it from
blocking GAL4
• This allows GAL1,7, and 10
to be transcribed at high
levels
•
Mosaic Analysis with a Repressible Cell Marker
MARCM
Heat Shock(HS)
driven Flp enzyme
causes recombination
between homologous
FRT sites during
mitosis
• This derepresses a
tissue-specific Gal4
transgene by
recombining away
Gal80 repressor
• Leading to clones of
cells positively
marked by the
membrane marker
CD8-GFP
Lee and Luo, Neuron 1999
MARCM can label a single neuron in the fly brain
CD8-GFP
nc82
Two types of regulation that we
will not have time to discuss
• Regulatory proteins that act by modifying chromatin
structure to allow transcription
– Nucleosomes block the promoters of most inactive genes.
– Some proteins such as the SWI-SWF complex in yeast act
by modifying chromatin structure to give the basal
transcription machinery greater access to promoters.
• Regulation of mRNA accumulation by controlling
mRNA stability
– Length of poly-A tail
– RNA-binding proteins that protect RNA from degradation
Regulation of Gene Expression in
Eukaryotes
Chapter 21
Eukaryotic gene regulation is different
from regulation of prokaryotic genes
• Complex chromosome structure
• Multiple chromosomes
• Spatial & temporal separation of
transcription and translation
• Transcripts processed in nucleus
• mRNA lasts longer
• Must have translational controls
• Multicellular with many cell
types
Eukaryotic genes have promoters and enhancers
enhancer
promoter
Eukaryotic genes vary in the number and arrangement of
controlling elements
Mutations in the Promoter Region Can
Drastically Alter Transcription
Overview of initiation of eukaryotic transcription
•TBP of TFIID binds
~20bp
•IIA & IIB Bind
•RNA Polymerase II, IIF
binds
•IIE,IIH, IIJ bind
•Promoter clearance
occurs
•basal transcription ensues
Enhancers control chromatin structure and transcription rate
•Different from promoters because….
•Position need not be fixed
•Orientation is not critical
•Introduced enhancers speed up transcription
How do they work?
Enhancers cause loops in the DNA which allow TFs
to interact with transcription complex
Stabilizes transcription complex
Transcription factors have functional domains that permit
binding to DNA and others that facilitate binding to protein in
transcription complex
True activator transcription factors include….
Helix-turn-helix
and…..
Zinc Fingers
and finally…...
Leucine Zippers
Antirepressor Transcription factors
• Change chromatin
structure to allow
polymerase
binding
•Several
processes
involved
ATP dependent remodeling (SWI/SNF)
Histone modification by Histone Acetyltransferase
Enzymes (HAT)
•Acetylation of
basic amino acids
of histone lessen
interaction with
DNA
•Deacetylases
(HD) reverse this
Methylation of CG doublets regulates gene expression
•Expression high when methylation low
•tissue specific
Alternative splicing of mRNA occurs in different tissues
•E.g. preprotachykin gene product
Controlling mRNA stability
• General or cell specific
• Stability sequences
• Instability sequences (e.g. AUUUA on fos
mRNA)
• Address sequences
• Translational controls
Translation controls can be used to regulate final gene products
•Tubulin gene regulated by tubulin subunit conc. in
cytoplasm (autoregulation)