Cell and Molecular Biology 48: Lac Operon

Strains of Escherichia coli

Three strains received:
- Temperature sensitive Lac mutant (lacZ_{ts})
- Temperature sensitive His mutant (hisA_{ts})
- Temperature sensitive RNA polymerase mutant (rpoB_{ts})
Distinguishing strains by growth on selective media is possible.
Conditions where all strains can grow:
- Minimal medium plus glucose at 30°C (permissive temperature)
- Rich medium at 30°C, allowing separate growth and plating.
Experimental Setup:
- Six plates suggested for controls.
  - Two plates with minimal media plus glucose.
  - Two plates with minimal media plus glucose and histidine.
  - Two plates with minimal media with lactose instead of glucose.
- Half grown at 30°C, half at 37°C.
Growth Results:
- hisA_{ts}: Grows on Glc 30°C, Glc, His 30°C/37°C, Lac 30°C, but not Glc 37°C and Lac 37°C.
- lacZ_{ts}: Grows on Glc 30°C/37°C, Glc, His 30°C/37°C, Lac 30°C, but not Lac 37°C.
- rpoB_{ts}: Grows on Glc 30°C, Glc, His 30°C, Lac 30°C, but not Glc 37°C, Glc, His 37°C and Lac 37°C.

Definition: A group of genes under the control of the same promoter.
Common in prokaryotes.
Allows genes to be regulated together.
Polycistronic mRNA: Encodes more than one protein.
Example: rRNA in E. coli (16S, 23S, and 5S), transcribed from a single promoter and cleaved.
Not all genes code for protein.

Making RNA and protein is energy costly.
Genes are switched on and off based on environmental conditions.
Bacteria in minimal medium must synthesize all nutrients (except glucose) from inorganic components.
In rich medium, genes for making available amino acids are switched off.

Not constitutively transcribed.
E. coli prefers glucose, then other carbon sources.
Machinery for other carbon sources is only activated when needed.
Diauxic Growth:
- Two growth phases.
- Carbon sources used consecutively, not simultaneously.
- Lag phase occurs when glucose is depleted.
- E. coli turns on lac genes and starts growing again.
Minimal Growth Requirements for E. coli hisam supF aziR: minimal medium with glucose and histidine, azide.

lacY: encodes β-galactoside-permease
lacZ: encodes β-galactosidase
- Gal \ Glc \ \beta-1,4
lacA: encodes galactoside acetyl-transferase
- Transfers an acetyl group to galactosides and glucosides.
- Function may be detoxification.

Normally turned OFF.
E. coli avoids wasting energy on utilizing rare sugars when not present.
When the operator (Op) is bound by a repressor, the operon is turned OFF.
Allolactose as Inducer:
- Molecule that turns genes on by disabling the repressor.
- Isomer of lactose with a β-1,6 bond.
- Lactose \ \beta-1,4 bond.
- Allolactose formation can be catalyzed by β-galactosidase (LacZ) even when the operon is repressed at a basal level.
When glucose is depleted and lactose is present:
- Allolactose binds the repressor, turning the operon ON.
- RNA Polymerase binds the promoter to produce LacZ, LacY, and LacA mRNA proteins.
De-repression:
- Uninduced cells have <10 LacZ molecules.
- Fully induced cells have >3000 LacZ molecules.

Glucose suppresses lac operon activation.
Catabolite Activator Protein (CAP) enhances transcription.
- CAP protein interacts with cAMP and binds to DNA.
- Enhances transcription ~50 times.
- RNA polymerase interacts favorably with CAP proteins.
Glucose inhibits cAMP production.
- cAMP is made by adenylate cyclase, which is inhibited by glucose.
- When allolactose is bound by repressor, the operon is turned ON, but slow transcription occurs without CAP protein.
Catabolite Repression:
- Glucose inhibits adenylate cyclase, resulting in no cAMP.
- The lac operon is transcribed slowly.
- This mechanism applies to other sugars as well.

The lac promoter can control other genes.
Useful for producing proteins for biotechnology (e.g., insulin) and for studying protein structure and function.
Desirable Characteristics of the lac Promoter
- Strong promoter for high mRNA production.
- Regulated expression to avoid constant expression due to energy cost.
- However, growing on lactose is inconvenient; glucose is preferred.
IPTG as a Lactose Analogue
- IPTG is a lactose analogue used for induction of expression.
- chemical \ structure \ of \ lactose \ and \ IPTG
Variants of the lac promoter used today are insensitive to glucose, allowing growth on glucose.
Growth slows down on induction.

Operons are groups of genes regulated together and transcribed from a single promoter.
The lac operon is normally repressed; lactose presence leads to de-repression.
Catabolite repression: bacteria prefer glucose and will not express genes for other carbon sources if glucose is present.
The lac promoter is widely used in biotechnology.