Lac and Trp Operons

Operon

An operating unit of functionally related genes that are regulated together, commonly found in prokaryotes.

Prokaryotic gene organisation

Most prokaryotic genes are organised into operons.

Function of an operon

Coordinates the expression of functionally related genes, for example genes involved in metabolic activity.

Polycistronic operon

An operon in which one mRNA molecule codes for more than one protein.

Polycistronic mRNA

mRNA that contains coding sequences for multiple proteins.

Polar expression

Expression in an operon is often polar, meaning more protein is produced from the first gene.

Bacterial gene expression

Transcription and translation are coupled in bacteria.

Coupled transcription and translation

Translation can begin while the mRNA is still being transcribed in bacteria.

Lac operon

The bacterial operon containing genes involved in lactose catabolism.

Lactose

A disaccharide found in milk, composed of glucose and galactose.

Lactose catabolism

The breakdown of lactose into simpler molecules.

Catabolism

The breakdown or degradation of molecules.

Lactose hydrolysis

The breakdown of lactose into glucose and galactose.

Lactose operon

Another name for the lac operon.

Inducible operon

An operon whose expression is switched on in response to an inducer.

Lac operon regulation

The lac operon is induced by lactose and repressed by the LacI repressor.

Inducer

A molecule that switches on gene expression, often by inactivating a repressor.

Lactose as an inducer

Lactose induces lac operon expression through the formation of allolactose.

De-repression

The removal or inactivation of a repressor, allowing transcription to occur.

Jacques Monod

Scientist associated with the discovery of the lac operon regulatory mechanism.

François Jacob

Scientist associated with the discovery of the lac operon regulatory mechanism.

Nobel Prize 1965

Award associated with Jacob and Monod for work on genetic control of enzyme and virus synthesis.

Plac

The promoter of the lac operon; the binding site for RNA polymerase.

Olac

The operator of the lac operon; the binding site for the LacI repressor.

PlacI

The promoter of the lacI repressor gene.

lacI

The gene encoding the repressor protein that regulates the lac operon.

LacI repressor

The protein that binds the lac operator and inhibits transcription initiation.

lacZ

The lac operon gene encoding beta-galactosidase.

lacY

The lac operon gene encoding lactose permease.

lacA

The lac operon gene encoding thiogalactoside transacetylase.

lacZ, lacY and lacA

Structural genes of the lac operon involved in lactose utilisation.

Structural genes

Genes that encode proteins or functional products.

Beta-galactosidase

The enzyme encoded by lacZ that hydrolyses lactose into glucose and galactose.

Lactose permease

The membrane transport protein encoded by lacY that facilitates lactose uptake.

Thiogalactoside transacetylase

The enzyme encoded by lacA.

Basal level of lacZ expression

A low level of lacZ expression that occurs even when the lac operon is repressed.

Basal expression

Low background gene expression occurring even under conditions where expression is largely repressed.

Allolactose

A lactose-derived molecule that binds the LacI repressor and causes it to dissociate from the operator.

Function of allolactose

Acts as the natural inducer of the lac operon by binding to LacI.

IPTG

A laboratory chemical that binds the LacI repressor and causes it to dissociate from the operator.

Function of IPTG

Artificially induces lac operon expression by inactivating the repressor.

Lac operon without lactose

The lac operon is repressed because the LacI repressor binds to the operator.

Constitutive expression of lacI

The lacI gene is expressed continuously, producing the LacI repressor.

What happens when LacI binds Olac?

The repressor inhibits transcription initiation by preventing RNA polymerase from proceeding effectively from the promoter.

What happens when allolactose binds LacI?

The repressor changes conformation and dissociates from the operator.

Lac operon with lactose

The inducer allolactose binds LacI, removing repression and allowing transcription.

Why is the lac operon inducible?

Because its expression is switched on in the presence of lactose.

Negative regulation of the lac operon

LacI binds the operator and prevents transcription initiation.

Negative feedback control

The lac operon is described as being under negative feedback control because repressor activity is regulated by lactose.

DNA-protein interaction

An interaction between DNA and a protein, such as LacI binding to the operator.

Conformational change

A change in a protein's three-dimensional shape that can alter its activity or interactions.

Cis-acting element

A regulatory DNA sequence that regulates only DNA to which it is physically joined.

Trans-acting factor

A diffusible regulatory factor, usually a protein, that can regulate genes at different DNA locations.

Example of a trans-acting factor

LacI repressor.

Example of a cis-acting element

The lac operon promoter or operator.

Meaning of cis

Next to, alongside or adjacent.

Meaning of trans

Across or between.

Cis-acting regulation

Regulation restricted to the DNA molecule containing the regulatory sequence.

Trans-acting regulation

A factor can regulate genes anywhere, including separate DNA molecules.

Cis-acting mutations

Mutations in regulatory DNA elements, such as the operator.

Trans-acting mutations

Mutations that alter regulatory proteins, such as the LacI repressor.

Dominant mutations in the lecture

Cis-acting mutations are described as dominant.

Recessive mutations in the lecture

Trans-acting mutations are mostly described as recessive.

lacOc mutation

A mutation in the lac operator that prevents normal repressor binding and causes constitutive expression.

Constitutive expression

Continuous expression of a gene regardless of normal regulatory signals.

Effect of lacOc mutation

The lac operon is constitutively expressed because the repressor cannot properly regulate the mutated operator.

Can lacOc be complemented by I+?

No. Introducing a functional I+ gene has no effect because the mutated operator acts in cis.

lacI- mutation

A mutation in the lacI gene that produces a non-functional repressor.

Effect of lacI- mutation

The lac operon becomes constitutively expressed because the repressor cannot properly inhibit transcription.

Can lacI- be complemented by I+?

Yes. A functional I+ gene can produce a working repressor that complements the mutation.

Complementation

The restoration of normal function by introducing a functional copy of a gene.

Why can trans-acting mutations be complemented?

A functional regulatory protein can diffuse and act on the relevant DNA.

Why can cis-acting mutations not be complemented?

The mutation affects a specific DNA regulatory site and cannot be repaired by supplying a functional protein.

Lac operon mutation summary

Regulatory DNA mutations are cis-acting and cannot be complemented; regulatory protein mutations are trans-acting and can be complemented.

Glucose

Glucose is the preferred energy source for bacteria.

Glucose preference

Bacteria preferentially use glucose over alternative carbon sources such as lactose.

Catabolite repression

The suppression of operons for alternative carbon sources by glucose.

Purpose of catabolite repression

Ensures preferential use of glucose before alternative carbon sources.

Alternative carbon sources

Carbon sources used when glucose is unavailable, such as lactose.

Diauxic growth

A growth pattern in which bacteria use one carbon source first and then switch to another.

Diauxic growth and glucose

Bacteria initially grow using glucose before switching to lactose when glucose is exhausted.

Glucose exhaustion

The point at which available glucose has been used up.

What happens after glucose is exhausted?

The lac operon can become activated if lactose is available.

Lag phase during diauxic growth

A temporary slowing of growth while bacteria activate the machinery needed to use lactose.

Beta-galactosidase during diauxic growth

Production increases after glucose is exhausted and the lac operon becomes activated.

What happens to alternative sugar operons when glucose is present?

Glucose represses several operons that allow the use of alternative carbon sources.

CRP

Catabolite Repressor Protein, a regulatory protein involved in catabolite repression.

CAP

Another name for CRP; stands for Catabolite Activator Protein.

cAMP

Cyclic adenosine monophosphate, a small molecule that binds CRP and permits its DNA binding.

Role of cAMP in lac operon regulation

cAMP binds CRP, allowing CRP to bind DNA and stimulate transcription.

CRP-cAMP complex

The complex formed when cAMP binds CRP, permitting DNA binding and transcriptional activation.

Role of CRP in the lac operon

Helps RNA polymerase bind and activate transcription of the lac operon.

What does RNA polymerase require for strong lac operon activation?

CRP bound to cAMP.

Relationship between glucose and cAMP

Cellular cAMP levels are controlled by glucose.

High glucose and cAMP

High glucose results in low cellular cAMP levels.

High glucose and the lac operon

The lac operon is switched off or has very low expression, even if lactose is available.

Low glucose and cAMP

Low glucose results in high cellular cAMP levels.

Low glucose and the lac operon

The lac operon can be activated if lactose is also present.

Why does high glucose suppress lac operon expression?

High glucose lowers cAMP, preventing effective CRP binding to DNA and transcriptional activation.

Why does low glucose favour lac operon expression?

Low glucose increases cAMP, allowing the CRP-cAMP complex to activate transcription if lactose is present.