Lac Operon
1
Lactose and Glucose
Much of the control of gene expression occurs at the
transcriptional level
Our understanding of transcriptional regulation comes from
studies of enzyme induction in E.coli
E. Coli exhibit an extremely sophisticated regulation of enzyme
Induction in response to changing environmental conditions.
The primary source of food for bacteria is glucose!
If both glucose and lactose are present together, glucose is
utilized first.
The organism will first breakdown glucose by turning on genes
for enzymes that metabolize glucose.
The enzymes required for lactose metabolism are shut off!
Once the glucose is completely metabolized, the genes
responsible for glucose metabolism are shut down. Then the
genes for the enzymes involved in lactose metabolism are turned
on.
How does a cell turn on and off these genes?
How does E. coli monitor the environment?
2
Gene Regulation
Questions were first addressed by studying genes controlling
lactose metabolism in E. coli
Lactose metabolism requires a b-galactosidase
When only lactose is present, the genes for lactose metabolism
are turned on.
When all the lactose is broken down, these enzyme are shut off
That is the genes coding for these enzymes are shut off
3
Gene Regulation
Cells– lactose
Cells + lactose
-galactosidase
-galactosidase
1
1000
very low level
induced
There are two ways you can visualize this occurring.
Lactose present
Lactose absent
gene ON
gene OFF
4
Gene Regulation
You can classify genes in a simple way in two classes
1) Structural
2) Regulatory
The structural genes are those that produce the enzyme required
for lactose metabolism
Permease
Galactosidase
Transacetylase
Allows transport of lactose into the cell
cleaves lactose into glucose and galactose
Unknown function
The regulatory loci determine whether transcription of the
structural genes will occur. They monitor and respond to
Environmental conditions (presence of lactose)
The loci that regulate lactose metabolic enzymes include
The repressor gene
The operator
The promoter
P
Promoter
O
Operator
Z
Y
Structural genes
A
5
LACTOSE (lac) OPERON
The operator is a specific DNA sequence to which the repressor
Binds
The promoter is a specific DNA sequence to which the RNA
Polymerase binds
By binding to the operator, the lac repressor prevents
transcription of the structural genes LacZ, LacY and LacA.
3 Structural genes
Permease
galactosidase
transacetylase
Y
Z
A
3 Regulatory elements
Operator
promoter
repressor
O
P
I
P
Repressor
6
The Lac operon
The structural genes and the regulatory elements form a functional
Genetic unit called the Lac Operon.
The repressor controls whether a RNA polymerase will transcribe
The lac operon genes
The repressor protein has a high affinity for binding the operator
DNA.
If repressor is bound to operator, the structural genes are not
Transcribed because the repressor physically blocks RNA
polymerase from transcribing the adjacent genes.
If repressor is not bound to the operator, the RNA polymerase
Can transcribe the structural genes
How is the operon regulated with respect to the environment?
When lactose is present in the media, the operon is ON and 7
When lactose is absent in the media, the operon is OFF.
The lac repressor
The repressor is the key element in regulating the operon
with respect to environmental conditions
The repressor has two functional sites
1) DNA binding site
2) Lactose binding site
When the repressor is bound to lactose, it no longer binds to
the operator DNA. Binding of lactose to the repressor
alters the conformation of the repressor protein so that
it no longer has a high affinity for the operator.
Lactose
Repressor
Repressor
No Lactose
Repressor
Repressor
The repressor is an example of an allosteric protein.
That is a protein that changes from one conformation to another
8
These changes alter the function of the protein.
No lactose present
9
Lactose present
10
11
Bacteria and Genetics
Jacob and Monod were the first to propose the operon model
of gene regulation following genetic analysis of E. coli
Griffiths; Fig 5-2, pg. 153
Genetically testing the lac operon model requires
complementation analysis which requires diploids
E. Coli are prokaryotes- by definition a haploid
12
Mechanism of DNA transfer
Partial diploids can be created in E.coli through the use of F’
Factors.
It involves Nonreciprocal (one way) transfer through F pilus
encoded by F factor
Jacob and Monod generated F’ factors carrying various parts
of the Lac Operon
These were used in complementation tests between other
factors in the operon
13
Griffiths; Fig 5-7, pg. 157
Pseudodiploid
a-
b-
A+
B+
a-
b-
c-
cc-
14
Analysis of lac operon
mutants using partial
diploids
Use of bacterial genetics to demonstrate
existence of promoters and repressors
Introduce F factors carrying mutations at the
Lac operon
Induce operon transcription
I
P
O
Z
Y
A
15
Analysis
I
P
O
Z
Repressor
Y
A
Repressor
Genotype
galactosidase(Z) Permease (Y)
no lac lac
no lac lac
---------------------------------------------------I+ P+O+Z+Y+
+
+
I- P+O+Z+Y+
+
+
+
+
Z+ and Y+ are coordinately expressed. Both are induced or not
induced together, because both are transcribed on a single mRNA
The I repressor is required to prevent Lac gene expression in the
absence of lactose.
The operon is negatively controlled. Its basal state is ON and
It must be actively turned OFF by binding of repressor to 16
operator
Analysis
I
P
O
Z
Repressor
A
Y
Repressor
Genotype
galactosidase(Z) Permease (Y)
no lac
lac
no lac
lac
---------------------------------------------------I- P+O+ Z+Y+
+
+
+
+
I- P+O+ Z+Y+/F(I+)
-
+
-
+
Experiment with partial diploid demonstrates whether a gene is
CIS or TRANS dominant (That is whether the gene product is
Diffusible or not)
I+
I-
P
O
Z
Y
A
17
Analysis
I
P
O
Repressor
Z
A
Y
Repressor
Genotype
galactosidase(Z) Permease (Y)
no lac
lac
no lac
lac
---------------------------------------------------I+ P+O+Z+Y+/
+
+
F(I+ P+O+Z-Y+)
I+ P+O+Z+Y+/
F(I+ P+O+Z+Y-)
-
+
-
+
I+ P+O+Z-Y+/
F(I+ P+O+Z+Y-)
-
+
-
+
Complementation of the structural genes still occurs
I
P
O
Z
Y
A
18
Analysis
I
P
O
Y
Z
A
mRNA
Z- cell
I
P
O
Z
Y
A
mRNA
Y- cell
I
P
O
Z
Y
A
mRNA
19
Is mutant
A new repressor mutant was discovered called Is
This mutant prevents induction of the lac structural genes
Genotype
galactosidase(Z)
no lac
lac
---------------------------------------------------I+P+O+Z+Y+
+
I- P+O+ Z+Y+
+
+
Is P+O+ Z+Y+
-
-
Is P+O+ Z+Y+/F(I+)
-
-
Is P+O+ Z+Y+/F(I-)
-
-
I+ P+O+ Z+Y+/F(Is)
-
-
The Is mutation is dominant to I+ and IHow can this mutant be explained?
20
Is mutant
A new repressor mutant was discovered called Is
This mutant prevents induction of the lac structural genes
Genotype
galactosidase(Z)
no lac
lac
---------------------------------------------------Is P+O+Z+Y+
-
-
The Is mutant eliminates the lactose binding site on the repressor
The repressor is always bound to the operator and blocks
transcription.
The presence of lactose in the media does not cause it to fall off
the DNA.
21
Is mutant
Genotype
galactosidase(Z)
no lac
lac
---------------------------------------------------I+P+O+Z+Y+/F(Is)
-
The Is mutant eliminates the lactose binding site on the repressor
The Is mutant is dominant to I+
How do you explain this?
I+
P
O
Z
Y
A
I+
I+
I+
Is
Is
Is
There is a 50:50 ratio of
Is to I+ in the cell
Is
22
Is mutant
I+
P
O
Z
Y
A
I+
I+
I+
Is
Is
Is
There is a 50:50 ratio of
Is to I+ in the cell
Is
So at random you would expect 50% of the operators to be
bound by I+ and 50% by Is
If this were the case then you should see half the operons
being induced and galactosidase being made (50%)
This does not occur
The reason is as soon as lactose binds and releases the normal
repressor (I+), the Is mutant repressor irreversibly binds the
operator.
23
LacO
The operon model also proposes a direct interaction between the
Repressor and a specific DNA site on the DNA (operator)
The repressor binds the operator and physically prevents RNA
Polymerase from transcribing the structural genes
Repressor
Repressor
I
P
O
Z
Y
A
24
Lac Operator mutants
Genotype
galactosidase(Z) Permease (Y)
no lac lac
no lac lac
---------------------------------------------------I+P+O+Z+Y+
+
+
I+P+Oc Z+Y+
+
+
+
+
I+P+Oc Z+Y+/F(O+)
+
+
+
+
I+P+O+Z+Y+/F(Oc)
-
+
-
+
I-P+O+Z+Y+
+
+
+
+
Results of #3 suggests that Oc is dominant to O+
Results of #4 suggests that O+ is dominant to Oc
The allele of O (Oc or O+) this is directly adjacent to the
structural genes (in the cis configuration) determines the
expression pattern of the structural genes. This is called Cisdominance in contrast to the Trans- dominance pattern observed
for the I gene.
The Oc mutation only disrupts transcription of genes directly
adjacent to it (on the same DNA). It has no effect on the
transcription of genes on other chromosomes
25
The repressor (I) is a diffusible protein (Trans dominant).
The operator (O) is a DNA sequence that binds the repressor.
LacO
The Oc mutation only affects the expression of those genes
Adjacent and on the same chromosome. It has no effect on
The expression of genes on other chromosomes
I+
I
I
P
O
Z
Y
A
No Txn
P
Oc
Z
Y
Oc
A
Txn
In general genes that exhibit Trans-dominance produce
a diffusable protein and regions of the DNA to which
proteins binds exhibit a Cis-dominance.
26
Catabolite repression
Catabolite Repression of the lac operonWhen we introduced the subject of regulation of gene
expression, I gave an example of the response of E. coli when
placed in a media containing both glucose and lactose. The
induction of the lac operon does not occur until all the glucose has
been metabolized. E. coli contains a mechanism that favors
glucose uptake first. Once the glucose is gone, the lactose will be
utilized.
This result is not predicted by the lac operon model we
presented. According to the model, lactose is solely responsible
for the regulation of this operon. Input from other environmental
factors, such as the presence of glucose, could not be accounted
for.
Eventually it was realized that some catalytic breakdown product
of glucose prevented the activation of the lac operon even in the
presence of lactose.
This effect was termed: Catabolite repression.
27
CAP
In addition to negative regulation by the lac repressor, the lac operon
also required positive regulation. That is, a factor was needed for
specific activation of the operon.
The specific activator was called CAP ( Catabolic activator protein)
produced by the gene crp (not part of the lac operon).
CAP forms a complex with cyclic AMP (cAMP). This complex is capable
of activating the lac operon.
There is an inverse relationship between the amount of glucose in the
cell and the amount of cAMP/CAP.
High glucose levels result in Low cAMP/CAP levels
Low glucose levels result in High cAMP/CAP levels
When glucose levels are high, little cAMP will be available to form the
CAP-cAMP activating complex of the lac-operon
In the positive control by the cAMP/CAP complex, the lac operon
becomes responsive to levels of glucose in the media.
28
CAP mediated activation of the lac operon
How does the cAMP/CAP complex regulate the lac operon.
This complex binds to a region of the promoter and is required for
RNA polymerase binding.
The lac operon possesses both negative regulation
(inducer/repressor) and a positive control system (cAMP/CAP
system).
29
Mendocino 2004 Prop H-Ban use of genetically modified organisms
DNA or Deoxyribonucleic acid means a complex protein that is
present in every cell of an organism and is the blueprint for
30
the organisms development