Lac operon, trp operon, Operon, structure and regulation

Concept 11.1 Several Strategies Are Used to Regulate Gene Expression
Gene expression is tightly regulated.
Gene expression may be modified to counteract environmental
changes, or gene expression may change to alter function in the
cell.
Constitutive proteins are actively expressed all the time.
Inducible genes are expressed only when their proteins are
needed by the cell. It is based on a feedback mechanism
• Negative regulation—a repressor protein prevents transcription
• Positive regulation—an activator protein binds to stimulate
transcription

Figure 11.1 Potential Points for the Regulation of Gene Expression
Points of Potential Regulation of
Genes
Transcription
Processing of mRNA
Translation
Post-translational

Control of Gene Expression
DNA -------------> RNA ------------> protein
transcriptional
control
translational
control
Operon: (prokaryotes) several adjacent genes regulated together and
coding for proteins involved in a common process
Operator: Repressor binding site
Promoter: transcriptional activation site
repressor gene promoter operator gene A gene B gene C

Gene Expression is precisely regulated
Gene Expression is precisely regulated
Maintain stable conditions -hormones
To perform cellular functions, keratin in skin, hemoglobin in
RBC’s

Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after
Transcription
Three ways to regulate mRNA translation:
• Inhibition of translation with miRNAs
• Modification of the 5′ cap end of mRNA can be modified—if cap is
unmodified mRNA is not translated.
• Repressor proteins can block translation directly—translational repressors

Figure 11.2 Positive and Negative Regulation
Gene expression begins at the
Promoter where RNA
Polymerase binds to initiate
transcription
A gene cluster with a single
promoter is an operon
An operator is a short stretch
of DNA near the promoter that
controls transcription of the
structural genes.
General Operon Model

Operons
An operon is a group of
genes that are transcribed
at the same time.
They usually control an
important biochemical
process.
They are only found in
prokaryotes. Jacob, Monod & L
woff
© 2016 Paul Billiet ODWS

12.2 Operon Structure
Operon: promoter + additional sequences that control
transcription (operator) + structure genes
Regulator gene: DNA sequence encoding products that
affect the operon function, but are not part of the operon

Two types of regulatory
proteins—transcription factors
—control whether a gene is
active.
Repressors are
Negative
Activators are Positive,
AKA Inducers

Negative and Positive Control; Inducible and
Repressible Operons
Inducible operons: Transcription is usually off
and needs to be turned on.
Repressible operons: Transcription is normally
on and needs to be turned off.

Negative and Positive Control; Inducible
Negative inducible operons: The control at the operator
site is negative. Molecule binding is to the operator,
inhibiting transcription. Such operons are usually off and
need to be turned on, so the transcription is inducible.
Inducer: small molecule that turns on the transcription

How does it work?
The lac operon is under two forms of control; positive and
negative control.
Negative control occurs when the binding of a protein prevents
an event. The brake on the car
Positive control is when the binding causes the event. The
accelerator on the car.

Negative and Positive Control; Repressible
Operons
• Negative repressible operons: The control at the operator
site is negative. But such transcription is usually on and
needs to be turned off, so the transcription is repressible.
• Corepressor: a small molecule that binds to the repressor
and makes it capable of binding to the operator to turn off
transcription.

Negative and Positive Control; Inducible and
Repressible Operons
• Positive inducible operon
• Positive repressible operon

The lac Operon of Escherichia coli
A negative inducible operon
Lactose metabolism
Regulation of the lac operon
Inducer: allolactose
– lacI: repressor encoding gene
– lacP: operon promoter
– lacO: operon operator

Example of gene control
Prokaryotes conserve energy by making proteins only
when needed.
In a rapidly changing environment, the most efficient
gene regulation is at the level of transcription.
E. coli must adapt quickly to food supply changes in
Glucose or lactose.

Many Prokaryotic Genes Are Regulated in Operons
Generally, inducible (positive) systems control catabolic
pathways—turned on when substrate is available
Repressible systems (negative) control anabolic
pathways—turned on until product becomes
excessive

Lac Operon encodes for the lactose enzymes
Lac Operon is an Inducible operon (Positive)—turned off
unless needed
If lactose is present and glucose is low, E. coli synthesizes
enzymes.
If lactose is absent, synthesis stops.
A compound that induces protein synthesis (transcription)
is an inducer.

Uptake and metabolism of lactose by E. coli involve
three proteins:
-galactoside permease—a carrier protein that moves
sugar into the cell
-galactosidase—an enzyme that hydrolyses lactose
-galactoside transacetylase—transfers acetyl groups
to certain -galactosides
If E. coli is grown with no lactose present, no enzymes
for lactose conversion are produced.

Lactose
O
OH
H
OH
OH
CH2OH
lactose
O H
OH
OH
OH
CH2OH
galactose glucose
O reducing end

Two kinds of factors
Jacob and Monod were able to identify 2 different
acting factors; cis and trans acting factors.
Cis acting factors only control transcription on the same
piece of DNA
Trans acting factors influence transcription on other
pieces of DNA

Lac Operon Inducible Gene operon Model Active Repressor-no
Transcription
Gene
region

The lac Operon
 The lac operon consists of three genes each involved
in processing the sugar lactose
 One of them is the gene for the enzyme β-
galactosidase
 This enzyme hydrolyses lactose into glucose and
galactose.

Gene Activation Inactivate Repressor Transcription takes place

 Lac genes.
◦ a. b-galactosidase is lacZ.
◦ b. Permease is lacY.
◦ c. Transacetylase is lacA.
◦ d. The genes are tightly linked in the order: lacZ-lacY-lacA.
 The three genes are transcribed on one polycistronic (polygenic)
RNA. Premature translation termination prevents this by reducing
translation of the downstream genes
The genes and their product

The lac operon is only transcribed when a -galactoside
predominates in the cell:
• In the presence of a -galactoside, the repressor
detaches and allows RNA polymerase to initiate
transcription.
• A repressor protein is normally bound to the operator,
which blocks transcription.
The key to this regulatory system is the repressor
protein.

Adapting to the environment
E. coli can use either glucose, which is a
monosaccharide, or lactose, which is a disaccharide
However, lactose needs to be hydrolysed (digested)
first
So the bacterium prefers to use glucose when it can.

The Lac Operon
When the repressor is bound to the operator
region it masks the -10 region of the promoter
and prevents the binding of RNA polymerase
to the promoter  NO transcription.
The actual structural genes are lac Z (beta
galactosidase), Y (lac permease) and A (lac
acetylase).
These three genes are all transcribed in one long
mRNA, known as a polycistronic mRNA.

The control of gene expression
Each cell in the human contains all the genetic
material for the growth and development of a
human
Some of these genes will be need to be
expressed all the time
These are the genes that are involved in of vital
biochemical processes such as respiration
Other genes are not expressed all the time
They are switched on an off at need.

 E. coli expresses genes for glucose metabolism constitutively, but the genes for metabolizing
other sugars are regulated in a “sugar specific” way. Presence of the sugar stimulates synthesis of
the proteins needed.
 Lactose is a disaccharide (glucose + galactose). If lactose is E. coli’s sole carbon source, three
genes are expressed:
◦ a. -galactosidase has two functions:
 i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and
glucose is metabolized by constitutively produced enzymes.
 ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation
of the lac operon.
◦ b. Lactose permease (M protein) is required for transport of lactose across the
cytoplasmic membrane.
◦ c. -Galactoside transacetylase transfers an acetyl group from acetyl-CoA to -
galactoside for reasons that are not understood.
Lactose as a Carbon Source for E. coli

The lac operon shows coordinate induction:
 a. In glucose medium, E. coli normally has very low
levels of the lac gene products.
 B. When lactose is the sole carbon source, levels of the
three enzymes increase coordinately (simultaneously)
about a thousandfold.
 i. Allolactose is the inducer molecule.
 ii. The mRNA for the enzymes has a short half-life.
When lactose is gone, lac transcription stops, and
enzyme levels drop rapidly.
The lac operon

1. When lactose is absent
A repressor protein is continuously synthesised. It sits on a
sequence of DNA just in front of the lac operon, the Operator
site
The repressor protein blocks the Promoter site where the RNA
polymerase settles before it starts transcribing
Regulator gene
lac operon
Operator site
z y a
DNA
I
O
Repressor
protein
RNA polymerase
Blocked

2. When lactose is present
A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
z y a
DNA
I O

2. When lactose is present
A small amount of a sugar allolactose is formed within the
bacterial cell. This fits onto the repressor protein at another
active site (allosteric site)
This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the operator
site. RNA polymerase can now reach its promoter site
Promotor site
z y a
DNA
I O

3. When both glucose and lactose are present
This explains how the lac operon is transcribed
only when lactose is present
BUT….. this does not explain why the operon is
not transcribed when both glucose and lactose
are present.

When glucose and lactose are present RNA polymerase
can sit on the promoter site but it is unstable and it
keeps falling off
Promotor site
z y a
DNA
I O
Repressor
protein removed
RNA polymerase

4. When glucose is absent and lactose is present
Another protein is needed, an activator protein. This stabilises
RNA polymerase.
The activator protein only works when glucose is absent
In this way E. coli only makes enzymes to metabolise other
sugars in the absence of glucose.
Promotor site
z y a
DNA
I O
Transcription
Activator protein
steadies the RNA
polymerase

1 2 3
O
P
R
NO Transcription of Lac operon genes
1 2 3
O
P
R
Transcription of Lac operon genes
repressor
Inactive repressor
Can’t link with operator, so
the Lac operon genes
expression switches on
In presence of Lactose
and absence of
glucose
RNA-
polymerase
1 2 3
Single
mRNA
1 2 3
Translation to 3
separate
enzymes
- Permease
- ß- galactosidase
- Galactoside transacetylase
Glucose
blocked
Operator
unblocked
Inducer
(allolactose)
Small molecule
formed from
lactose
1) Binding of repressor to the operator
switches operator off. (negative Control)
2) In presence of lactose, few molecules enter
the cell and act as inducer. (positive Control)
repressor

lactose
Permease
Transport Lac
across the Pl.m.
- ß- galactosidase
- Galactoside
transacetylase
- Galactose
- Glucose
E. Coli plasma
membrane
Lactose
allolacto
se
(Inducer)
Few
molecules
enter the cell
& form
allolactose
Lac operon of E. Coli :
Catabolizes the disaccharide
lactose into glucose (in presence
of lactose & absence of glucose).

Summary
Carbohydrates Activator
protein
Repressor
protein
RNA
polymerase
lac Operon
+ GLUCOSE
+ LACTOSE
Not bound
to DNA
Lifted off
operator site
Keeps falling
off promoter
site
No
transcription
+ GLUCOSE
- LACTOSE
Not bound
to DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
- LACTOSE
Bound to
DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
+ LACTOSE
Bound to
DNA
Lifted off
operator site
Sits on the
promoter site
Transcription

1) Negative Control.
•- Inhibit the activity of Lac operon as
economical process in presence of
glucose.
•-The controlling -element is the
repressor protein that switches the
transcription off.
•- in presence of glucose:
bacteria produces repressor
binds to operator inactive
operator turn transcription
off.
2) Positive Control.
•- activation of lactose catabolism.
•- Pomoter of Lac operon has low affinity
for RNA-polymerase, although the
repressor protein is inactive by
allolactose.
•- Activation of Lac operon;
- Takes place by CAP (catabolic
activator protein).
- CAP is inactive, becomes active as it
combines with cAMP (co-activator) to
form CAP- cAMP- complex.
- cAMP is regulated by glucose (it is
inversely proportional to glucose
concentration).
glucose cAMP
Types of transcriptional control of
Types of transcriptional control of
Lac Operon
Lac Operon

Four situations are possible
1. When glucose is present and lactose is absent the
E. coli does not produce β-galactosidase.
2. When glucose is present and lactose is present the
E. coli does not produce β-galactosidase.
3. When glucose is absent and lactose is absent the E.
coli does not produce β-galactosidase.
4. When glucose is absent and lactose is present the
E. coli does produce β-galactosidase.

Mutated Lac Operon & Areas
what if …?
I-
no repressor Lac operon turned on
Oc
repressor can’t bind Lac operon turned on
to operator
Is
allolactose can’t bind Lac operon turned off
to repressor
I-d
repressor can’t bind Lac operon turned on
to operator

(a) Under conditions of high glucose,
a glucose breakdown product
inhibits the enzyme adenylate
cyclase, preventing the conversion of
ATP into cAMP.
(b) As E. coli becomes starved for
glucose, there is no breakdown
product, and therefore adenylate
cyclase is active and cAMP is formed.
(c) When cAMP (a hunger signal) is
present, it acts as an allosteric
effector, complexing with the CAP
dimer.
(d) The cAMP-CAP complex (not CAP
alone) acts as an activator of lac
operon transcription by binding to a
region within the lac promoter. (CAP
= catabolite activator protein; cAMP
= cyclic adenosine monophosphate)
Catabolite control of the lac operon
CAP sites are also present in other promoters.
cAMP-CAP is a global catabolite gene
activator.

What is cAMP?
This is a compound synthesised from ATP by adenylyl
cyclase which acts in both eukaryotes and prokaryotes
as a second messenger.
In this situation cAMP ↓ when glucose enters the
bacterial cell
This causes the cAMP associated with CAP to come off
 CAP without cAMP then comes off the DNA

Positive control and catabolite repression
Catabolite repression: using glucose when
available, and repressing the metabolite of other
sugars.
The positive effect is activated by catabolite
activator protein (CAP). cAMP is bound to CAP,
together CAP–cAMP complex binds to a site
slightly upstream from the lac gene promoter.

• One last gene encodes catabolite activator
protein (CAP)
• Three domains
• DNA binding
• Multimerization
• Allosteric
• Forms a homodimer
• Cyclic AMP (cAMP) is the ligand that induces
conformational change increased DNA binding
• cAMP is produced only when glucose is not
present

• The lac operon structure
• A multi-part regulatory region
• CAP binding site – bound by catabolite activator protein
• Promoter – bound by RNA polymerase
• Operator – bound by lac repressor
Bacterial Gene Regulation

Positive control and catabolite repression
cAMP―adenosine-3′,5′-cyclic monophosphate
The concentration of cAMP is inversely
proportional to the level of available glucose.

Cooperative binding of cAMP-CAP and RNAP on the lac promoter
cAMP-CAP contacts the -subunits of RNAP and enhances the
binding of RNAP to the promoter.

Positive and negative regulation of the lac operon

Positive Control
This is exerted by a protein known as the
Catabolite Activator Protein (CAP)
This protein binds to a site on the DNA within
the promoter region and increases the rate of
RNA polymerase binding; hence transcription
initiation.
It only does this when complexed to cAMP.

 When both glucose and lactose are in the medium, E.
coli preferentially uses glucose, due to catabolite
repression.
◦ a. Glucose metabolism greatly reduces cAMP
levels in the cell.
◦ b. The CAP–cAMP level drops and is insufficient
to maintain high transcription of the lac genes.
◦ c. Even when allolactose has removed the
repressor protein from the operator, lac gene
transcription is at very low levels without CAP–cAMP
complex bound to the CAP site.
◦ d. Experimental evidence supports this model.
Adding cAMP to cells restored transcription of the
lac operon, even when glucose was present.
Both glucose and lactose situation

Lac Operon under Different Cell Conditions
what if…?
no glu, high lac high cAMP, high allolac high transcription
high glu, no lac low cAMP, low allolac no transcription
no glu, no lac high cAMP, no allolac no transcription
some glu & lac low cAMP, some allolac
not enhanced, induced
low transcription

What happens when lactose enters the cell?
A small amount of it is converted to
allolactose (this is NOT a substrate for
beta galactosidase)
The allolactose then binds to the
repressor
The repressor: allolactose complex
dissociates from the operator
Transcription can begin.

Putting it all together
When lactose is present the
repressor is off the operator.
When glucose is present the CAP
is off the promoter.

Mutations in lac
Regulator gene mutations: lacI−
leads to constitutive
transcription of three structure genes.
• lacI+
is dominant over lacI−
and is trans acting. A single copy o
lacI+
brings about normal regulation of lac operon.
• lacI+
lacZ−
/ lacI−
lacZ+
produce fully functional β-galactosidase.

Mutations in lac
Regulator gene mutations: lacI−
leads to constitutive
transcription of three structure genes.
• lacI+
is dominant over lacI−
and is trans acting. A single copy of lacI+
brings
about normal regulation of lac operon.
• lacI+
lacZ−
/ lacI−
lacZ+
produce fully functional β-galactosidase.

Mutations in lac
• Operator mutations: lacOc
:
C = constitutive
• lacOc
is dominant over lacO+
,
which is cis acting.
• lacI+
lacO+
Z–
/ lacI+
lacOc
lacZ+
produce fully functional β-
galactosidase constitutively.

Mutations in lac
• Promoter mutations
• lacP−
: cis acting
• lacI+
lacP−
lacZ+
/ lacI+
lacP+
lacZ−
fails to produce
functional
β-galactosidase.

Terms
Negative and Positive Control
Cis and Trans acting factors
Global and Specific regulation.

Different Scenarios
1. Lactose (+) and glucose (-)
2. Lactose (+) and glucose (+)
3. Lactose (-) and glucose (+)
4. Lactose (-) and glucose (-)

Scenario 1
Lactose (+) glucose (-)
Some of the Lactose entering the cell via
the few lac permease transporters
available has been converted to
allolactose and has resulted in the
removal of the repressor from the
operator.

Scenario 1
The promoter is now unmasked and RNA
polymerase can now bind and initiate
transcription.
However it won’t do this very frequently
without the help of the cAMP-CAP
bound to the activation site.

Scenario 1
This protein complex binding puts a 90o
kink in
the DNA and interacts with the alpha
subunit of RNA polymerase.
Without the cAMP:CAP the lac promoter is a
weak promoter varying significantly from
the consensus sequence at -10 and -35. The
combination of the two controls means
beta gal and lac permease are transcribed
at high levels.

Scenario 1
Repressor with allolactose bound is now off the
operator and the CAP with cAMP bound is
on the DNA in a region upstream from the
promoter.
The RNA pol can bind to the promoter and the
CAP:cAMP can act positively to enhance the
frequency of transcription initiation.
Brake off accelerator on

Scenario 2
Lactose (+) and glucose (+)
The repressor is off the operator but the
CAP protein (without cAMP) is not
bound to the DNA so initiation only
occurs at a low rate  little
transcription.
Brake off but accelerator off also

Scenario 3
Lactose (-) and glucose (+)
The repressor is bound to the operator
and the CAP (without cAMP) is not
bound to the DNA. Very little
transcription of the lac operon genes
is happening now.
Brake on and accelerator off…not going
anywhere fast

Scenario 4
Lactose (-) and glucose (-)
The cell is starving! The repressor is on the
operator but the cAMP CAP is on the DNA.
If the repressor is bound there is no
transcription  RNA polymerase has no
access.
Brake on and accelerator on (but you have run
out of petrol!)

Global Regulation
A factor can influence the transcription of
a number of genes scattered
throughout the genome e.g. CAP.
A number of genes which encode
catabolic enzymes involved in
carbohydrate metabolism are
controlled by CAP e.g. arabinose
operon

Specific Regulation
A factor can influence the transcription of
a few genes downstream e.g. lac
repressor
Only the next string of genes in the
operon, which are transcribed to a
single polycistronic meassenger RNA
will be affected.

• Wait a minute?!?!
• If no lactose is present, transcription is shut down
• No permease is available to allow lactose in
• And even if it got in, it wouldn’t be metabolized and no
allolactose would be produced to release the repressor
from the operator
• How does transcription EVER start?
• Leaky transcription
• Binding of the repressor is reversible
• Sometimes it just falls off, allowing a very low level of
transcription and low levels of permease and -
galactosidase in the cell

trp operon
Five structural genes A-E
The promoter and the operator regions are upstream from trpE
Between the promoter-operator and trpE is a short leader
region, trpL
With in trpL is an attenuator site that plays an important role in
the regulation of the trp operon

The trp Operon of E. coli
• A negative repressible operon
• Five structural genes
• trpE, trpD, trpC, trpB, and trpA – five enzymes together
convert chorismate to typtophane.

• The trp operon
• Tryptophan is an essential amino acid that can
be synthesized by the cell
• But, why bother if tryptophan is already
present?
• The trp operon is repressible, meaning it’s
usually on but can be turned off
• Furthermore, it can be fine tuned to match the
needs of the cell  a process called
attenuation

Regulation of the trp operon
The regulatory gene for the trp operon is trpR
The product of trpR is an corepressor protein, which is an
inactive repressor that alone cannot bind to the operator
When tryptophan is abundant within the cell, it interacts
with the corepressor and converts it to an active repressor
The active repressor binds to the operator and prevents the
initiation of transcription of the trp operon
 In the presence of excess tryptophan a hairpin loop is formed
that behaves as a terminator structure
 As a result the tryptophan enzymes are not produced

• The trp operon structure
• A multi-part regulatory region and five structural genes
• Structural genes
• trpA-E  enzymes involved in the anabolism (building molecules
• trpR  elsewhere, encodes trp Repressor

• trp repressor protein has a similar structure
but works the opposite way of lac repressor
• Three domains
• DNA binding
• Multimerization
• Allosteric
• Forms a homodimer
• Tryptophan is the ligand (corepressor) that
induces conformational change  increased
DNA binding

Figure 11.9 The trp Operon: A Repressible System

The trp Operon of Escherichia coli
• A negative repressible operon
• Five structural genes
• trpE, trpD, trpC, trpB, and trpA―five enzymes together
convert chorismate to tryptophane.

Figure 11.6 Two Ways to Regulate a Metabolic Pathway

a.There are five structural genes, trpA through trpE.
b.The promoter and operator are upstream from the trpE gene.
c.Between trpE and the promoter-operator is trpL, the leader region. Within
trpL is the attenuator region (att).
d.The trp operon spans about 7 kb. The operon produces a polygenic
transcript with five structural genes for tryptophan biosynthesis.
Gene Organization of the Tryptophan Biosynthesis Genes

Concept 11.3 Eukaryotic Genes Are Regulated by Transcription Factors
and DNA Changes
Transcription factors act at eukaryotic promoters.
Each promoter contains a core promoter sequence
where RNA polymerase binds.
TATA box is a common core promoter sequence—rich
in A-T base pairs.
Only after general transcription factors (TF) bind to the
core promoter, can RNA polymerase II bind and
initiate transcription.

If amino acids are available in the medium, E. coli will import them
rather than make them, and the genes for amino acid biosynthesis
are repressed. When amino acids are absent, the genes are
expressed and biosynthesis occurs.
Unlike the inducible lac operon, the trp operon is repressible.
Generally, anabolic pathways are repressed when the end product
is available.
The trp Operon of E. coli

Regulation of the trp operon
The second regulatory mechanism occur
when tryptophan is limited
Under severe tryptophan starvation the
trp genes are expressed
If tryptophan is scarce an antiterminator
hairpin loop is formed
As a result transcription is allowed to
proceed past the involved DNA
sequence and the entire mRNA is
produced

Two mechanisms regulate expression of the trp
operon:
a. Repressor–operator interaction.
b. Transcription termination.
Regulation of the trp Operon

Repressor Gene Trp Operon
A repressible operon is switched off when its
repressor is bound to its operator.
However, the repressor only binds in the
presence of a co-repressor.
The co-repressor causes the repressor to change
shape in order to bind to the promoter and
inhibit transcription.
Tryptophan functions as its own co-repressor,
binding to the repressor of the trp operon.

When tryptophan is limited, transcription is also controlled by
attenuation.
 a. Attenuation produces only short (140 bp) transcripts
that do not encode structural proteins.
 b. Termination occurs at the attenuator site within the
trpL region.
 c. The proportion of attenuated transcripts to full-length
ones is related to tryptophan levels, with more attenuated
transcripts as the tryptophan concentration increases.
 d. Attenuation can reduce trp operon transcription 8- to
10-fold. Together, repression and attenuation regulate trp
gene expression over a 560- to 700-fold range.
Expression of the trp Operon in the Presence of
Low Concentrations of Tryptophan

The molecular model for attenuation:
 a. Translation of the trpL gene produces a short polypeptide. Near the
stop codon are two tryptophan codons.
 b. Within the leader mRNA are four regions that can form secondary
structures by complementary base-pairing
 i. Pairing of sequences 1 and 2 creates a transcription pause signal.
 Ii. Pairing of sequences 3 and 4 is a transcription termination signal (a rho-
independent terminator).
 Iii. Pairing of 2 and 3 is an antitermination signal, and so transcription will
continue.
Molecular Model for Attenuation

Tight coupling of transcription and translation in prokaryotes makes
control by attenuation possible.
 i. RNA polymerase pauses when regions 1 and 2 base-pair just after they are
synthesized
 ii. During the pause, a ribosome loads onto the mRNA and begins translation of
the leader peptide. Ribosome position is key to attenuation:
 (1) When tryptophan (Trp) is scarce:
 (a) Trp–tRNAs are unavailable, and the ribosome stalls at the Trp codons in
the leader sequence, covering attenuator region 1.
 (b) When the ribosome is stalled in attenuator region 1, it cannot base-pair
with region 2. Instead, region 2 pairs with region 3 when it is synthesized.
 (c) If region 3 is paired with region 2, it is unable to pair with region 4 when
it is synthesized. Without the region 3–4 terminator, transcription
continues through the structural genes.

(2) When Trp is abundant:
 (a) The ribosome continues translating the leader peptide, ending
in region 2. This prevents region 2 from pairing with region 3,
leaving 3 available to pair with region 4.
 (b) Pairing of regions 3 and 4 creates a rho-independent
terminator known at the attenuator. Transcription ends before the
structural genes are reached.

Trp Operon Controlled by Attenuation
trpR P O 1 2 3 4 trpE trpD trp C trp B trpA
attenuator
Leader
•attenuation can form
under certain conditions
•base-pairing can occur
between
1 - 2 2 - 3 3 - 4
Attenuation

High levels of Tryptophan in Cell
•transcription & translation occur simultaneously in Prokaryotes
•leader transcript (1) has 2 trp codons (UGGUGG)
•ribosomes moves fast along transcript
•stem-loop 3 - 4 forms, poly Us after
•early termination of transcription,
translation stops
(only leader peptide forms
- has no function)

Low Levels of Tryptophan in Cell
•ribosome stalls at UGGUGG in leader transcript (1)
•stem-loop 2 - 3 forms, no poly U after
•transcription continues

Low Levels of other Amino Acids
•ribosome stalls way early
•stem-loops 1-2 & 3-4 form, poly U after
•early termination of transcription

• The lac operon structure
• A multi-part regulatory region
• Promoter – bound by RNA polymerase
• Operator – bound by trp repressor
• Attenuator – we’ll get to that

• Let’s put it all together
• Three scenarios exist
• Tryptophan + as needed
• Tryptophan + but low
• Tryptophan -
What does the cell ‘want’ to
do in each case and how
is it accomplished?

• Tryptophan +
• The cell ‘wants’: to use use the available tryptophan, so why bother
transcribing genes to metabolize it?
• The cell accomplishes this by: shutting down the trp operon

• Tryptophan -
• The cell ‘wants’: tryptophan and needs to manufacture it for itself
• The cell accomplishes this by: activating the trp operon

• Tryptophan +/-
• The cell ‘wants’: some tryptophan but not too much  fine tune
production
• The cell accomplishes this by: attenuating (taper off) the trp operon
• As tryptophan increases in the cell  production decreases
• As tryptophan decreases in the cell  production increases
• The result is a steady-state, or homeostasis
• Attenuation involves the leader strand (trpL) segment of the trp
operon mRNA
• Somehow, increased tryptophan availability results in the premature
termination of trp operon transcription

• Tryptophan +/-
• As trp increases rate of trp operon transcription decreases
• Of the transcripts that are produced, more and more consist only of
the first 140 nt from the 5’ end of trpL
trp =
Full length transcripts
Partial (inviable) transcripts

• Tryptophan +/-
• trpL contains
• Four repeated DNA sequences
• Can form stem-loop structures
• A region that codes for a 14 AA polypeptide
• Two back-to-back codons code for tryptophan

• Tryptophan +/-
• The four repeats can form three, mutually exclusive structures
• 2-3 loop = antitermination loop
• 3-4 loop = termination loop

• Tryptophan +/-
• 3-4 loop = termination loop
• Remember WAY back in chapter 8?
• Intrinsic termination
• If 3-4 loop forms, transcription of trp is stopped

Attenuation in the trp Operon of E. coli
• Four regions of the long 5′ UTR (leader) region of
trpE mRNA
• When tryptophan is low, region 2 binds to region
3, which prevents the binding of region 3 and
region 4, and transcription continues.

Promoter Promoter
DNA trpR
Regulatory
gene
RNA
polymerase
mRNA
3
5
Protein Inactive
repressor
Tryptophan absent, repressor inactive, operon on
mRNA 5
trpE trpD trpC trpB trpA
Operator
Start codonStop codon
trp operon
Genes of operon
E
Polypeptides that make up
enzymes for tryptophan synthesis
D C B A
ressible system
essible system
bolic pathway (synthesis of amino acid tryptophan)
bolic pathway (synthesis of amino acid tryptophan)

DNA
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Active
repressor
No RNA made

Synthesis
of enzymes
Switch
transcription on
Turn operator
on
Inactive
repressor
co-repressor -
rep. complex
Switch operator
off
Turn transcription
off
Stop enzyme
synthesis
Tryptophan
level
(tryptophan
act as co-
repressor)
Decrease
Increase

• Tryptophan +/-
• 2-3 loop = antitermination loop
• 3-4 loop cannot form  transcription continues
• How does the cell control this in such a way to encourage or
discourage trp expression?
• As tryptophan increases in the cell  transcription decreases
• As tryptophan decreases in the cell  transcription increases

• Tryptophan +/-
• Depends on whether or not the ribosome is stalled in region 1
• If tryptophan is readily available, ribosome has no trouble filling the
need for two sequential tryptophans during translation
• Ribosome moves rapidly through 1 and covers 2, preventing 2 from
interacting with 3
• 3-4 termination loop forms, halting transcription of the full operon

• Tryptophan +/-
• If tryptophan is readily available, ribosome has no trouble filling the
• Ribosome moves rapidly through 1 and covers 2, preventing 2 from
interacting with 3
• 3-4 termination loop forms, halting transcription of the full operon

• Tryptophan +/-
• Depends on whether or not the ribosome is stalled in region 1
• If tryptophan is in short supply, ribosome has difficulty filling the
• Ribosome stalls at 1, allowing 2 to interact with 3
• 2-3 antitermination loop forms, allowing transcription of the full
operon

• Tryptophan +/-
• If tryptophan is in short supply, ribosome has difficulty
filling the need for two sequential tryptophans during
translation
• Ribosome stalls at 1, allowing 2 to interact with 3
• 2-3 antitermination loop forms, allowing transcription of
the full operon

• Tryptophan +/-
• http://highered.mheducation.com/sites/dl/free/0072835125/126997/
animation28.html

a. There are five structural genes, trpA through trpE.
b. The promoter and operator are upstream from the trpE gene.
c. Between trpE and the promoter-operator is trpL, the leader region.
Within trpL is the attenuator region (att).
d. The trp operon spans about 7 kb. The operon produces a polygenic
transcript with five structural genes for tryptophan biosynthesis.
Gene Organization of the Tryptophan Biosynthesis Genes

16.4 Some Operons Regulate Transcription Through
Attenuation, the Premature Termination of Transcription
• Attenuation: affects the continuation of
transcription, not its initiation. This action terminates
the transcription before it reaches the structural
genes.

Attenuation in the trp Operon of E. coli
• Four regions of the long 5′ UTR (leader) region of
trpE mRNA
• When tryptophan is high, region 1 binds to region
2, which leads to the binding of region 3 and
region 4, terminating transcription prematurely.

Types of Transcriptional Control in Prokaryotes
Types of Transcriptional Control in Prokaryotes
NEGATIVE CONTROL
NEGATIVE CONTROL
1)
1) Inducible genes
Inducible genes
- Represser protein alone
- Represser protein alone
lactose repressor alone
lactose repressor alone
-
- Represser protein + inducer
Represser protein + inducer
lactose repressor + allolactose
lactose repressor + allolactose
2)
2) Repressible genes
Repressible genes
- Repressor protein alone
- Repressor protein alone
Tryptophan repressor alone
Tryptophan repressor alone
-
- Repressor + corepressor
Repressor + corepressor
Tryptophan repr. + tryptophan
Tryptophan repr. + tryptophan
Active represser "turns off" regulated gene.
Active represser "turns off" regulated gene.
Lactose operon not transcribed.
Lactose operon not transcribed.
Inactive repressor/inducer complex fails to
Inactive repressor/inducer complex fails to
"turn off" regulated gene(s).
"turn off" regulated gene(s).
Lactose operon transcribed.
Lactose operon transcribed.
Inactive represser fails to "turn off" regulated
Inactive represser fails to "turn off" regulated
gene(s).
gene(s).
Typrtophan operon transcribed.
Typrtophan operon transcribed.
Active repressor-corepressor complex "turns
Active repressor-corepressor complex "turns
off" regulated gene(s).
off" regulated gene(s).
Tryptophan operon not transcribed.
Tryptophan operon not transcribed.
POSITIVE CONTROL
POSITIVE CONTROL
- Activator protein alone
- Activator protein alone
CAP alone
CAP alone
- Activator protein + coactivator
- Activator protein + coactivator
CAP + cAMP
CAP + cAMP
Activator alone cannot stimulate
Activator alone cannot stimulate
transcription of regulated gene(s).
transcription of regulated gene(s).
Transcription of lactose operon not stimulated
Transcription of lactose operon not stimulated
Functional activator-coactivator complex
Functional activator-coactivator complex
stimulates transcription of regulated gene(s).
stimulates transcription of regulated gene(s).
Transcription of lactose operon stimulated.
Transcription of lactose operon stimulated.

Lac operon, trp operon, Operon, structure and regulation

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