BSCI223: Lac Operon

What is the overall function of the lac operon?

It encodes enzymes necessary for metabolism of lactose

What type of mRNA is produced by the lac operon?

Polycistronic mRNA of 3 genes

What are the 3 genes?

lacZ, lacY, lacA

What does lacZ encode for?

Encodes LacZ, or β-galactosidase (β-gal)

What does lacY encode for?

Encodes LacY, or Lactose Permease

What does lacA encode for?

Encodes LacA, or β-galactosidase transacetylase

What is the function of LacY (Permease)?

It transports lactose into the cell

What reaction does LacZ (β-galactosidase) catalyze?

It breaks down Lactose into Glucose and Galactose

What are three ways bacteria control gene expression?

• Use of different sigma factors to control many genes

• Control via use of repressors and inducers

• Control via small molecule activation

Is the lac operon inducible or repressible?

Highly regulated inducible operon (genes only transcribed when needed)

When is the lac operon turned on and off?

• Turned on in the presence of lactose

• Turned off in the absence of lactose

What happens when LacI (repressor) is bound to the operator site?

Transcription of the lac operon does not occur

What is a Repressor?

Protein that prevents gene expression by binding to specific DNA sequence (operator)

How does LacI block transcription?

It bends the DNA and prevents access of RNA Polymerase (RNAP) to the lac promoter

What happens to the lac operon if glucose becomes scarce and lactose is added?

Lactose induces the operon (lactose reverses repression)

What is the mechanism of induction by lactose?

The repressor protein binds the modified lactose molecule, changes conformation, and cannot bind to the operator site

What is a Inducer?

A substrate that induces gene expression of genes coding for enzymes necessary for its own metabolism

What is an Apoinducer?

A protein that enhances gene expression; it binds a region near the promoter and enhances RNA Pol binding

What is CAP and what does it bind to?

Catabolite Activator Protein (an apoinducer); it binds the promoter when in complex with cyclic AMP (cAMP)

How do glucose levels affect cAMP concentration?

• In presence of glucose → Low concentration of cAMP

• In absence of glucose → High concentration of cAMP

When glucose is low, what happens to cAMP?

High cAMP; cAMP forms complex with CAP, transcription enhanced

When glucose is high, what happens to cAMP?

Low cAMP; No cAMP-CAP complex formed

What two systems act together in activating the Lac operon?

• Control via inducer (lactose binding to repressor)

• Control via small molecule activation (cAMP binding to CAP)

Will the lac operon be activated if there is no glucose (high cAMP) but also no lactose?

No, the lac operon will not be activated if there is no lactose, even when cAMP levels are high

What is the transcription level of lacZ with "- Lactose"?

No lacZ transcription

What is the transcription level of lacZ with "+ Lactose" and "+ Glucose"?

Low lacZ transcription

What is the transcription level of lacZ with "+ Lactose" and "- Glucose"?

High lacZ transcription

What is Glucose Effect?

E. coli uses glucose first; it only switches to lactose after glucose runs out

What is Catabolite Repression?

The regulatory mechanism by which glucose inhibits expression of operons needed to metabolize alternative carbon sources (ie. lactose)

What is one consequence of Catabolite repression?

Diauxic growth; growth on glucose → pause when glucose runs out → second growth phase once the lactose-utilizing enzymes are induced

What does this graph show?

Diauxic growth

Why does glucose show a steeper slope in log phase growth compared to lactose?

Glucose metabolism is more efficient

What is CAP?

Catabolite activator protein

What does CAP do?

It is a positive regulator that helps RNA polymerase bind the lac promoter

What turns CAP on?

cAMP; cAMP is high when glucose is low

When does CAP bind DNA?

When glucose is absent (cAMP is high)

When is the lac operon ON?

Lactose present (LacI off) AND glucose absent (CAP on)

What is X-gal used for?

It acts as a substrate for β-gal and an indicator

What is the result when β-Galactosidase acts on X-Gal?

It produces a blue precipitate

Is the trp operon inducible or repressible?

Repressible operon

What is the function of the trp operon?

Encodes enzymes necessary for tryptophan biosynthesis

What is the state of the Trp repressor when Tryptophan is absent?

Inactive repressor; operon is ON

How does Tryptophan affect the Trp repressor?

The repressor protein is inactive until bound by tryptophan. Once bound, it becomes an active repressor and the operon is OFF

Why does the cell shut down Trp synthesis genes when Trp is present?

Too much Trp is toxic