Transcript Document

Lac Operon
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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?
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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
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Gene Regulation
Cells– lactose
Cells + lactose
-galactosidase
-galactosidase
1
1000
low level
induced
There are two ways you can visualize this occurring.
Lactose present
Lactose absent
gene ON
gene OFF
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Gene Regulation
You can classify genes in a simple way in two classes
1)
2)
The structural genes are those that produce the enzyme required
for lactose metabolism
The regulatory elements 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
P
O
Z
Y
A
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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.
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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
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.
The repressor is an example of an allosteric protein.
That is a protein that changes from one conformation to another
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These changes alter the function of the protein.
No lactose present
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Lactose present
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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
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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
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Griffiths; Fig 5-7, pg. 157
Pseudodiploid
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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
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Analysis
I
Repressor
P
O
Z
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
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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
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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
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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?
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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
---------------------------------------------------IsP+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.
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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?
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Is mutant
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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
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LacO mutants
Genotype
galactosidase(Z) Permease (Y)
no lac lac
no lac lac
---------------------------------------------------I+P+O+Z+Y+
I+P+OcZ+Y+
I+P+OcZ+Y+/F(O+)
I+P+O+Z+Y+/F(Oc)
I-P+O+Z+Y+
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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
In general genes that exhibit Trans-dominance produce
a diffusable protein and regions of the DNA to which
proteins binds exhibit a Cis-dominance.
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Catabolite repression
Catabolite Repression of the lac operon-
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CAP
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CAP mediated activation of the lac operon
How does the cAMP/CAP complex regulate the lac operon.
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