The lac operon – An inducible operon

• Genes are either cis (coupling) acting– Genes affect those adjacent to themselves– Operator and structural genes

Or

• trans (repulsion) acting– Genes affect other genes that are not adjacent to

themselves– Implies the action of a diffusible product– Regulator genes

Trans (repulsion) acting genes•Genes that affect other genes•Are not adjacent to the genes they control–Regulator genes

Implies the action of a diffusible product

Genes that are cis (coupling) acting

•Genes affect genes adjacent to themselves

Operator and structural genes

The lac operon – An inducible operon

• Bacteria are monoploid – one copy of each gene

• Can make bacteria partially diploid (two copies of a gene)– Place another copy of the gene(s) on a plasmid

(F’)

– E.coli lac I mutant with a plasmid carrying a functional repressor gene will be designated: I - O + Z +/F’ I +

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I +

I + O c Z +/F’ I +

I + O + Z +/ F’ I s

I s O c Z +/F’ I +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I +

I + O c Z +/F’ I +

I + O + Z +/ F’ I s

I s O c Z +/F’ I +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I +

I + O c Z +/F’ I +

I + O + Z +/ F’ I s

I s O c Z +/F’ I +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I + + -

I + O c Z +/F’ I +

I + O + Z +/ F’ I s

I s O c Z +/F’ I +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I + + -

I + O c Z +/F’ I + + +

I + O + Z +/ F’ I s

I s O c Z +/F’ I +

I s O c Z -/F’ I -

plasmid

E. Coli genome

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I + + -

I + O c Z +/F’ I + + +

I + O + Z +/ F’ I s - -

I s O c Z +/F’ I +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I + + -

I + O c Z +/F’ I + + +

I + O + Z +/ F’ I s - -

I s O c Z +/F’ I + + +

I s O c Z -/F’ I -

The lac operon – An inducible operon

beta-galtosidase activity

beta-galactosidase

activity

Genotype Lactose present Lactose absent

I + O + Z + + -

I - O + Z +/F’ I + + -

I + O c Z +/F’ I + + +

I + O + Z +/ F’ I s - -

I s O c Z +/F’ I + + +

I s O c Z -/F’ I - - -

The lac operon – An inducible operon

In F’ plasmid, an entire lac operon can be supplied. For example,

I + O + Z - Y +/F’ I - O + Z + Y -

What are the beta-galactosidase and permease activities with and without lactose in the E. coli strain with the above genotype?

The lac operon

• If E. coli cells are provided with both glucose and lactose, it preferentially metabolizes glucose rather than lactose. How do E. coli cells accomplish this?

• Positive control and catabolite repression

Positive Control and Catabolite Repression

New Players:Cyclic AMP (cAMP)

CAP (catabolite activator protein)

High glucose low cAMP

Low glucose high cAMP

Positive Control and Catabolite Repression

High glucose low cAMP

Low glucose high cAMP

Positive Control and Catabolite Repression

High glucose low cAMP

Low glucose high cAMP

Positive Control and Catabolite Repression

The cAMP-CAP complex interacts with the promoter

(neither can bind the promoter of lac operon by themselves)

CAP = Catabolite Activator Protein

Facilitates the attachment of the RNA polymerase to the promoter – acts as an activator

Must be a cAMP-CAP complex to bind to the promoter

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)

(low)

No transcription

The cAMP-CAP complex is not formed (low cAMP), so CAP does not bind to the promoter

In addition, there is no inducer (lactose), so the active repressor is bound to the operator, and therefore the RNA polymerase cannot bind and transcribe the Lac operon genes

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

2. Glucose and lactose both present (+)

The cAMP-CAP complex does not form (low cAMP)

The inducer (lactose) is present and inactivates the repressor•There is some transcription, but it is inefficient (no cAMP-CAP complex)

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

2. Glucose and lactose both present (+)– Very little transcription

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

2. Glucose and lactose both present (+)– Very little transcription

3. Glucose absent (-), but lactose present (+)

The cAMP-CAP complex does form (high cAMP), facilitating the attachment of the RNA polymerase to the promoter

The RNA polymerase is effectively attached to the promoter, enhancing transcription

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

2. Glucose and lactose both present (+)– Very little transcription

3. Glucose absent (-), but lactose present (+) – A lot of transcription

Positive Control and Catabolite Repression

What would happen to the lac operon when:

1. Glucose present (+), but lactose absent (-)– No transcription

2. Glucose and lactose both present (+)– Very little transcription

3. Glucose absent (-), but lactose present (+)– A lot of transcription

4. Glucose and lactose both absent (-)

No inducer (no lactose) No transcriptionRepressor stays bound to the operator

cAMP pairs with CAP and can attach to the promoter

Positive Control and Catabolite Repression

What would happen to the lac operon when:1. Glucose present (+), but lactose absent (-)

– No transcription2. Glucose and lactose both present (+)

– Very little transcription3. Glucose absent (-), but lactose present (+)

– A lot of transcription4. Glucose and lactose both absent (-)

– No transcription

Lac operon overview - animation

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html