Topic 20

Open reading frame (ORF)

A continuous sequence of DNA or RNA spanning from a start codon to a stop codon that encodes a single protein.

Monocistronic mRNA

An mRNA molecule that contains a single open reading frame and encodes one protein.

Polycistronic mRNA

An mRNA molecule carrying multiple open reading frames that are translated into separate proteins.

Operon

A cluster of adjacent genes controlled together by a single promoter and operator and transcribed as a unit.

Promoter

A specific DNA sequence where RNA polymerase binds to initiate transcription.

Operator (lacO)

A regulatory DNA sequence where the repressor protein binds to inhibit transcription.

Repressor

A regulatory protein that binds to an operator to block RNA polymerase from transcribing downstream genes.

Inducer

A specific small molecule that binds a regulatory protein to switch on gene expression.

Inducible expression

Gene expression that remains normally off or basal until an environmental cue activates it.

Repressible expression

Gene expression that remains continuously on until an environmental signal turns it off.

Constitutive expression

Continuous, unregulated gene transcription that proceeds regardless of environmental conditions.

Biological purpose of the lac operon

To coordinate the cellular import of lactose and its enzymatic breakdown for energy.

Product and function of lacZ

$\beta$-galactosidase, which cleaves lactose into glucose and galactose and isomerizes lactose into allolactose.

Product and function of lacY

Lactose permease, a membrane protein that facilitates the uptake of lactose into the bacterial cell.

Product and function of lacA

Thiogalactoside transacetylase, an enzyme that attaches acetyl groups to galactosides; nonessential for lactose consumption.

Product and expression of lacI

Encodes the Lac repressor protein and is continuously transcribed from its own independent promoter.

Allolactose

The physiological inducer of the lac operon that binds the Lac repressor to release it from the operator.

Role of glucose in lac operon regulation

Serves as the preferred carbon source; its presence prevents the low-glucose transcriptional boost.

Cyclic AMP (cAMP)

A signaling molecule that accumulates under glucose starvation to activate the catabolite activator protein.

Catabolite Activator Protein (CAP)

A regulatory protein that binds cAMP to form a complex that helps recruit RNA polymerase to the promoter.

CAP binding site

A specific regulatory DNA sequence upstream of the lac promoter where the CAP–cAMP complex attaches.

RNA polymerase

The primary enzyme responsible for synthesizing RNA from a DNA template during transcription.

Leaky expression

A baseline level of transcription that occurs when a repressor briefly falls off its operator.

Biological necessity of leaky lac operon expression

Maintains baseline amounts of permease to import lactose and $\beta$-galactosidase to convert it into the allolactose inducer.

Effect of the CAP–cAMP complex on transcription

Dramatically upregulates transcription by physically stabilizing and recruiting RNA polymerase to the promoter.

Mechanism of lac operon induction by lactose

Permease imports lactose $\rightarrow$ $\beta$-galactosidase generates allolactose $\rightarrow$ allolactose inhibits repressor $\rightarrow$ operator is freed $\rightarrow$ lacZYA are transcribed.

Mechanism of lac operon upregulation under low glucose

Glucose depletion elevates cAMP $\rightarrow$ cAMP binds CAP $\rightarrow$ CAP–cAMP binds the CAP site $\rightarrow$ RNA polymerase recruitment is enhanced.

lac operon expression level: No lactose and high glucose

Very low (basal/repressed): repressor remains bound to the operator, and no CAP–cAMP activation occurs.

lac operon expression level: No lactose and low glucose

Low: repressor is bound, though activated CAP–cAMP promotes occasional leaky transcription events.

lac operon expression level: Lactose present and high glucose

Moderate: inducer removes repressor from the operator, but no CAP–cAMP transcriptional boost occurs.

lac operon expression level: Lactose present and low glucose

High (fully induced): repressor is detached from the operator and CAP–cAMP actively recruits RNA polymerase.