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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 enzymeInduction 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 offThat is the genes coding for these enzymes are shut off

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Gene Regulation

There are two ways you can visualize this occurring.

Lactose present gene ONLactose absent gene OFF

Cells– lactose -galactosidase 1 low level

Cells + lactose -galactosidase 1000 induced

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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 requiredfor 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

Z Y AOP

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The operator is a specific DNA sequence to which the repressorBinds

The promoter is a specific DNA sequence to which the RNAPolymerase binds

By binding to the operator, the lac repressor prevents transcription of the structural genes LacZ, LacY and LacA.

LACTOSE (lac) OPERON

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The Lac operonThe structural genes and the regulatory elements form a functionalGenetic unit called the Lac Operon.

The repressor controls whether a RNA polymerase will transcribeThe lac operon genes

The repressor protein has a high affinity for binding the operatorDNA.

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 polymeraseCan 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 When lactose is absent in the media, the operon is OFF.

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The lac repressorThe 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 tothe operator DNA. Binding of lactose to the repressorAlters the conformation of the repressor protein so that itNo 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 anotherThese changes alter the function of the protein.

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No lactose present

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Lactose present

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Griffiths; Fig 5-2, pg. 153

Bacteria and Genetics

Jacob and Monod were the first to propose the operon model of gene regulation following genetic analysis of E. coli

Genetically testing the lac operon model requires complementation analysis which requires diploids

E. Coli are prokaryotes- by definition a haploid

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Griffiths; Fig 5-7, pg. 157

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 partsof the Lac Operon

These were used in complementation tests between other factors in the operon

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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 P O Z Y A

Repressor 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 operator

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Analysis

I P O Z Y A

Repressor Repressor

Experiment with partial diploid demonstrates whether a gene isCIS or TRANS dominant (That is whether the gene product isDiffusible or not)

I- P O Z Y AI+

Genotype galactosidase(Z) Permease (Y)no lac lac no lac lac

----------------------------------------------------I- P+O+ Z+Y+

I- P+O+ Z+Y+/F(I+)

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Analysis

I P O Z Y A

Repressor 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-)

I P O Z Y A

Complementation of the structural genes still occurs

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Analysis

I P O Z Y 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 IsThis 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 I-

How can this mutant be explained?

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Is mutant

A new repressor mutant was discovered called IsThis 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

The Is mutant eliminates the lactose binding site on the repressor

The Is mutant is dominant to I+

How do you explain this?

Genotype galactosidase(Z)no lac lac

----------------------------------------------------I+P+O+Z+Y+/F(Is)

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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

I P O Z Y A

Repressor

Repressor

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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.