MBIO 3410 - Lecture 6 (part 5: lac operon)

Lac operon (negative control)

The repressor blocks the RNA, preventing the gene from being transcribed, but when lactose comes in, an environmental signal binds to the repressor and causes it to unbind from the DNA, therefore allowing for transcription.

Inducer of the negative control mechanism of the lac operon

1.) The inducer is actually a side-product from lactose being cleaved and is not actually lactose itself

2.) This shows that genes are never really turned off because in order to cleave the lactose into allolactose (the side product), LacZ, which is encoded in the operon, is needed.

How can a gene have basal transcription if it’s being blocked by a repressor

Even when the RNA pol is being blocked by a repressor, there are times when the repressor will not be bound, because its affinity is not perfect, therefore allowing the RNA pol to pass and transcribe the gene in basal amounts.

Strength of lac operon promoter

1.) The lac operon actually has a relatively weak promoter

2.) Therefore, on top of the negative control mechanism, the lac operon also has a positive control mechanism, which is usually normal for most catabolizing genes

Positive control mechanism of the lac operon is for…

It prioritizes glucose rather than lactose

Components needed for the positive control mechanism

1.) CAP site (the activator binding site)

2.) CAP protein (catabolite activator protein, the activator)

3.) Cyclic AMP, the inducer that binds to the CAP protein and turns on the expression of the lac operon

Requirement for the CAP protein binding to the CAP site

It only binds to the CAP site if cAMP, the inducer, is bound to it

Dual control of the lac operon

Involves both positive and negative control mechanisms, both of which need to be met in order for transcription to proceed

Two conditions that need to be met in order to transcribe the lac operon

1.) Need cAMP (therefore need glucose to be absent)

2.) Allolactose binding to the lacI repressor (therefore need lactose to be present)

Positive control of the lac operon (turning on)

1.) cAMP binds to the CAP, and then the CAP-cAMP complex binds to the CAP site

2.) But glucose must be absenst

Negative control of the lac operon (turning on)

The lac repressor (LacI), must be absent from the operator, therefore lactose must be present

What happens to the lac promoter when glucose is absent

cAMP is high, allowing for the CAP-cAMP complex to bind to the CAP site, recruiting an RNA pol in the process, therefore transcription can proceed

What happens with the lac operon when glucose is present

1.) cAMP is low and therefore the CAP-cAMP complex cannot form and bind to the CAP site

2.) Therefore, the RNA pol is not recruited and transcription remains off

Why does the absent of glucose = high cAMP levels

1.) Glucose has a phospho-relay system that phosphorylates glucose as it enters the cell

2.) When glucose is absent, one of the enzymes in this relay system activates adenylate cyclase, which produces cAMP

Phospho-relay system of glucose

1.) Glucose is transported by the PTS (phosphoenolpyruvate phosphotransferase system)

2.) It passes the phosphate from one enzyme to the next, until it reaches the transmembrane protein that transports glucose into the cell, which passes the phosphate onto glucose to form glucose-6-phosphate

PTS when glucose is present

PTS is busy moving glucose into the cell

PTS when there is no glucose

The phosphates end up getting backed up, therefore allowing the phosphorylated EII enzyme to activate adenylate cyclase, the enzyme that produces cAMP

Adenylate cyclase

Turns ATP into cAMP

Prolonged absence of glucose

It generates a pool of cAMP, which is now available to interact with the CAP protein and turn on the transcription of the lactose degradation system.

Lac operon when no glucose + no lactose

CAP-cAMP binds to the cap site, but the repressor remains on the promoter since there is no lactose, therefore there is no transcription (except for basal transcription)

Lac operon when glucose + no lactose

CAP-cAMP does not bind to the CAP site and the repressor remains on the promoter, therefore there is no transcription and the basal transcription is even less than when the conditions were no glucose and no lactose were present.

Lac operon when lactose + no gluose

CAP-cAMP binds to the cap site and the repressor is not bound to the promoter since there IS lactose, therefore there is transcription (at the highest level), because both conditions are being met

Lac operon when yes glucose + yes lactose

CAP-cAMP does not bind to the CAP site and the repressor is not bound to the promoter, therefore there is no transcription (but it does have basal levels of transcription).

Complexity of the lac operon

It is an extremely complex system that involves multiple components, each of which are not only influenced by this system but also other systems, as they are constantly competing for these components.