Gene Regulation 2 & 3 - Negative & Positive regulation of the lactose operon Flashcards
1
Q
The lactose operon
A
1
Q
Lecture outcomes
A
- Draw a graph to illustrate usage of glucose and lactose
in E. coli - List the enzymes of the lactose operon and their
functions - Describe the lactose operon and its negative regulation,
using diagrams - Explain the terms inducer, inducible, on-off regulation
and diauxic growth
2
Q
A
2
Q
A
3
Q
A
3
Q
The Lactose (lac) Operon
A
An operon which is responsible for the transport and
metabolism of the sugar lactose in E. coli.
* Lactose is one of many organic molecules E. coli can
use as a carbon and energy source
* Glucose is the preferred C source for E. coli
* If we supply E. coli with both glucose and lactose, the
cells use the glucose until it is exhausted, stop growing
briefly, then start growing again using the lactose
3
Q
Growth of E. coli with glucose and lactose provided
A
- E. coli cells are grown on a medium containing both glucose and lactose,
and the bacterial density (number of cells/ml) is measured. Diauxic growth
is observed (cellular growth in two phases) - During the second lag phase the cells have been adjusting to the new
nutrient source by turning on the lac operon and accumulating the enzymes
needed to break down the lactose
4
Q
Growth of E. coli with glucose and lactose provided
A
4
Q
Enzymes needed for lactose metabolism in E. coli
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4
Q
The lactose operon is controlled by “on-off”
regulation
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This is an INDUCIBLE system
Lactose (strictly speaking, its derivative allolactose) is an INDUCER
of the production of the two enzymes
Inducer: small molecule that stimulates the synthesis of an
inducible protein
5
Q
The lactose operon is controlled by “on-off”
regulation photo
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6
Q
The reactions of β-galactosidase photo
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6
Q
The reactions of β-galactosidase
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- The main reaction catalysed by β-galactosidase is the hydrolysis of lactose
- It also catalyses a minor reaction that converts lactose to allolactose
- Allolactose acts as the inducer of β-galactosidase synthesis
7
Q
The lactose operon
A
8
Q
The lactose operon
A
This was the FIRST
operon discovered: Jacob
and Monod, work in
1950s, Nobel prize 1965
9
Q
A
- In the lac operon, the main operator is adjacent to the promoter
- The function of the lacA gene product (transacetylase) is not
well-understood – appears not to be required for lactose
catabolism - The lacI gene is upstream (5ꞌ) of the operon. It is transcribed
from its own promoter and translated separately, to give the
repressor protein
10
Q
Negative regulation of the lac operon
A
- When there is NO lactose in the surroundings, the enzymes
are not needed and are switched OFF - When the inducer lactose IS present, the enzymes are
needed and are switched ON - Inducer binds to repressor protein, alters repressor
conformation, prevents repressor binding to operator site
on DNA
11
Q
How does the lac repressor prevent
transcription of the lac operon?
A
- The lac repressor is a
tetramer with 2 identical
binding sites - The lac operator actually has
three sites: O1, O2, O3 - The repressor binds O1 and
either O2 or O3, forming a
DNA loop - The loop contains the -35 and
-10 binding sites recognised
by RNA polymerase - These sites are now
inaccessible to RNA
polymerase
11
Q
Negative regulation of the lac operon
A
11
Q
Comparison with negative regulation of the
tryptophan operon
A
- When there is NO tryptophan present on the
surroundings, the genes are switched ON - When there IS tryptophan present and it enters the
bacterial cell, the enzymes are no longer needed and
are switched OFF
* Difference is because the lac operon is a catabolic
(degradative) operon, while the trp operon is an
anabolic (biosynthetic) operon
11
Q
How does the lac repressor prevent
transcription of the lac operon?
A
12
Q
Negative regulation of the tryptophan operon
A
13
Q
Living cells need to make a lot of
decisions all the time!
A
14
Q
Negative regulation of the lactose operon:
overview
A
- Lactose metabolism in E. coli is carried out by two proteins, βgalactosidase and lactose permease
- The genes for these and transacetylase are clustered together and
transcribed from one promoter, giving a polycistronic mRNA, i.e.
they form an operon (lacZ, lacY, lacA) - Negative control of the lac operon works by a repressor protein
binding to the operator and preventing RNA polymerase from
binding to the promoter : no transcription - When lactose is present, its derivative allolactose acts as an
inducer by binding to the repressor causing it to dissociate from
the operator : transcription of the structural genes occurs
14
Negative versus positive regulation
15
Negative regulation of the lactose operon:
overview
* Lactose metabolism in E. coli is carried out by two proteins, βgalactosidase and lactose permease
* The genes for these and transacetylase are clustered together and
transcribed from one promoter, giving a polycistronic mRNA, i.e.
they form an operon (lacZ, lacY, lacA)
* Negative control of the lac operon works by a repressor protein
binding to the operator and preventing RNA polymerase from
binding to the promoter : no transcription*
* When lactose is present, its derivative allolactose* acts as an
inducer by binding to the repressor causing it to dissociate from
the operator : transcription of the structural genes occurs
*a simplified explanation. But (not for assessment) for those wondering why allolactose can be produced
in a cell which is un-induced, where it seems there should be no β-galactosidase present to make
allolactose : think about the low level of basal transcription always present even when an operon is “off” in
bacterial cells.
16
Negative regulation of the lactose operon:
overview
* Lactose metabolism in E. coli is carried out by two proteins, βgalactosidase and lactose permease
* The genes for these and transacetylase are clustered together and
transcribed from one promoter, giving a polycistronic mRNA, i.e.
they form an operon (lacZ, lacY, lacA)
* Negative control of the lac operon works by a repressor protein
binding to the operator and preventing RNA polymerase from
binding to the promoter : no transcription
* When lactose is present, its derivative allolactose acts as an
inducer by binding to the repressor causing it to dissociate from
the operator : transcription of the structural genes occurs
17
17
18
The lactose operon
18
Lecture Outcomes
* List the components important in positive regulation of the Lac operon
* Describe the positive regulation of the Lac operon, using diagrams
* List 3 regulatory DNA-binding proteins in bacteria
* Understand how proteins can bind to specific regions of DNA, and the roles
of some conformational changes caused by this binding
19
Positive regulation of the lac operon
Unlike the Trp operon, the Lac operon is under both positive and negative transcriptional
controls.
The components:
1. 1. The Lac promoter: is a relatively “weak” promoter. This means that RNA polymerase
recognises it rather poorly
2. 2. An activator protein called CAP*: is needed to help the RNA polymerase bind to the
promoter. It binds to DNA near the promoter
3. 3. Cyclic AMP (cAMP): is a small messenger molecule. CAP must bind to cAMP
before CAP can bind to DNA i.e. it is actually CAP-cAMP dimer that binds to DNA
*CAP = catabolite activator protein
Some textbooks call it CRP = cAMP receptor protein
19
Growth of E. coli with glucose and lactose
provided
20
Positive regulation of the lac operon
The function
* Glucose is the preferred sugar of E. coli (see graph of diauxic growth)
* E. coli cells keep the Lac operon inactive as long as glucose is
present
* The cells must have some way to sense the lack of glucose, and to
respond by activating transcription of the Lac operon (assuming
lactose is present)
20
Positive regulation of the lac operon
* When [glucose] drops, [cAMP] rises
* When cAMP levels rise, levels of CAP-cAMP dimer also rise (more cAMP
available to bind to CAP)
* CAP-cAMP dimer can bind to the Lac operon DNA and activate transcription
– So the Lac operon is activated only when glucose concentration is low and
a need arises to metabolise an alternative energy source, lactose
* Why is this positive regulation? Because there is an activator, not a repressor
protein. The binding of the CAP-cAMP protein to DNA activates transcription
21
Positive regulation of the lac operon
* cAMP responds to changes in glucose concentration
* It is an intracellular signalling molecule
* There is an inverse relationship between glucose levels and cAMP concentration
21
Growth of E. coli with glucose and lactose
provided
* E. coli cells are grown on a medium
containing both glucose and lactose,
and the bacterial density (number of
cells/ml) is measured. Diauxic growth is
observed (cellular growth in two
phases)
* During the second lag phase the cells
have been adjusting to the new nutrient
source by turning on the lac operon and
accumulating the enzymes needed to
break down the lactose
22
Positive regulation of the lac operon photo
23
Positive regulation of the lac operon
cAMP binds to CAP protein,
causing a conformational change
Allows CAP to bind to the CAP
site
The bound CAP-cAMP dimer
interacts with RNA polymerase
This greatly stimulates the rate of
transcription initiation.
24
Dual control of lac operon
The operon is
highly expressed
only when lactose
is present and
glucose is absent
24
Negative versus positive regulation
25
Dual control of lac operon
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26
DNA binding proteins
* The surface of the protein fits tightly against the surface of the specific DNA
region it recognises
* In most cases the protein inserts into the major groove of the DNA double helix
* The protein forms bonds with the bases (but does not disrupt the
complementary base pairing of A-T, G-C): bonds include
H bonds
ionic bonds
hydrophobic bonds
but NOT covalent bonds
* Overall the 20 or so contacts between protein and DNA make the binding:
very strong
highly specific
| HIH B
26
Dual control of lac operon
Negative regulation:
LacI binds to lac operon, preventing transcription
Allolactose binds to LacI → LacI dissociates from lac operon
Positive regulation:
CAP on its own cannot bind to lac operon
CAP-cAMP binds to lac operon, activating transcription
LacI is a repressor
Allolactose is an inducer
CAP is an activator
cAMP is an inducer
27
DNA binding proteins
28
Dual control of lac operon
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Negative versus positive regulation
30
DNA binding proteins
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DNA binding proteins
32
DNA binding proteins: repressor binding
to operator DNA
33
Conformational changes in DNA
CAP structure and DNA binding:
* CAP binds to the major groove in DNA
adjacent to the promoter of the lac operon.
* Upon binding, CAP-cAMP bends the DNA
by 90o
* This stimulates transcription of the operon
because it enhances the binding of RNA
polymerase to the DNA
34
Conformational changes in DNA
34
Conformational changes in DNA
35
Conformational changes in protein
36
Summary of conformational changes in
protein and DNA
* Binding of tryptophan to trp repressor changes repressor conformation
Enables repressor to bind tightly to operator DNA
* Binding of lactose to lac repressor changes repressor conformation
Prevents repressor binding to operator DNA
* Binding of lac repressor to operator forms loops in DNA
Prevents RNA polymerase from transcribing DNA
* Binding of CAP-cAMP dimer to CAP-binding site near the promoter causes the DNA to bend ~ 90°
The DNA bending allows RNA polymerase to bind to the promoter more efficiently, stimulates
transcription
37
Overall comments: bacterial gene
regulation
* Simple prokaryotic cells have quite complex systems of gene regulation
* Control of gene regulation is essential for life
* If all 4000 E. coli genes were active all of the time, the cell would be drained
of energy and unable to compete with more efficient organisms
* For bacteria, grouping functionally related genes together into operons, so
that they can be easily co-regulated, has been a very successful strategy
