Lac operon

Reading File

Finding Sources

Reading File

Loading Image

Here is a complete overview of the Lac Operon drawn from Harper's Illustrated Biochemistry (32nd Ed.) and Basic Medical Biochemistry (6th Ed.).

The Lac Operon

The lac operon of E. coli is the textbook model for coordinate prokaryotic gene regulation. It controls three enzymes for lactose catabolism using both negative and positive regulatory mechanisms.

1. Organization of the Lac Operon (~6 kb)

Lac operon map showing lacI, CRE, Promoter, Operator, lacZ, lacY, lacA and TSS

From Harper's Illustrated Biochemistry, 32nd Ed., Figure 38-2

The operon has two classes of elements:

Regulatory elements

Element	Function
lacI gene	Constitutively transcribed; encodes the LacI repressor protein
CRE (cAMP Response Element)	Binding site for CAP-cAMP complex (positive regulator)
Promoter	RNA polymerase binding site
Operator	LacI repressor binding site; overlaps the promoter; a 21-bp inverted palindrome

Structural genes

Gene	Protein	Function
lacZ	β-galactosidase	Cleaves lactose into glucose + galactose; also converts lactose → allolactose
lacY	Galactoside permease	Transports lactose into the cell
lacA	Thiogalactoside transacetylase	Acetylates β-galactosides (function less clear)

All three genes are transcribed into a single polycistronic mRNA with independent AUG/UAA codons, so each protein is translated separately (not cleaved from a precursor).

2. Structural Gene Products

Lac operon structural genes and protein products

From Basic Medical Biochemistry, 6th Ed., Figure 15.5

3. Regulatory Mechanisms

The lac operon is under dual control: negative regulation by the LacI repressor and positive regulation by CAP-cAMP.

A. Negative Regulation - The LacI Repressor

lacI is expressed constitutively, producing ~30 tetrameric LacI repressor molecules per cell
Each tetramer has four identical subunits (MW 38,000 each)
LacI binds the operator with very high affinity (Kd ~10⁻¹³ mol/L), occupying it >95% of the time
When bound, LacI blocks RNA polymerase from binding the promoter → no transcription of lacZ, lacY, lacA

Key detail on the operator: It contains a 21-bp inverted palindrome:

5'-AATTGT GAG C GATAACAATT
3'-TTAACACTCG C CTATTGTTAA

Two of the four repressor subunits contact the operator, making contacts in the major groove.

B. Induction by Allolactose

When lactose is present in the environment:

A small amount of lactose enters the cell via the low basal level of permease
β-galactosidase converts lactose → allolactose (the true inducer)
Allolactose binds LacI repressor → conformational change → affinity for operator drops ~10,000-fold (Kd ~10⁻⁹ mol/L)
LacI dissociates from operator → RNA polymerase binds promoter → transcription begins
Induction is rapid: lac mRNAs are maximally induced within ~5 min; β-galactosidase peaks within ~10 min
Under full induction: up to 5,000 β-galactosidase molecules per cell (~1000× basal level)

IPTG (isopropyl-thiogalactoside) is a classic "gratuitous inducer" - it induces the operon but is not hydrolyzed by β-galactosidase. Widely used in molecular biology for recombinant protein expression.

4. The Three States of the Lac Operon

Three scenarios for lac operon expression: no inducer, inducer + glucose, inducer + no glucose

From Harper's Illustrated Biochemistry, 32nd Ed., Figure 38-3

Condition	Repressor state	CAP-cAMP at CRE	Transcription
No lactose, glucose present	Active (bound to operator)	Absent (glucose → low cAMP)	OFF
Lactose + glucose	Inactive (allolactose bound)	Absent (glucose → low cAMP)	Very low
Lactose, no glucose	Inactive (allolactose bound)	Present (high cAMP → CAP active)	Fully ON

5. Positive Regulation - CAP and Catabolite Repression

When glucose is present, cells preferentially use it and suppress the lac operon:

High glucose → low cAMP levels
CAP (Catabolite Activator Protein, also called CRP) requires cAMP to bind the CRE site
Without CAP-cAMP at the CRE, RNA polymerase binds the promoter inefficiently → low transcription

When glucose is absent:

cAMP levels rise
cAMP binds CAP → conformational change → CAP-cAMP complex binds CRE upstream of promoter
This greatly stimulates RNA polymerase binding → high-level transcription

This phenomenon (suppression of other carbon-source operons in the presence of glucose) was originally called catabolite repression, but is actually a form of positive regulation.

6. Lac Operon Expression Summary

Glucose HIGH + Lactose absent  →  Repressor ON, CAP OFF  →  No expression
Glucose HIGH + Lactose present →  Repressor OFF, CAP OFF →  Minimal expression
Glucose LOW  + Lactose absent  →  Repressor ON, CAP ON   →  No expression
Glucose LOW  + Lactose present →  Repressor OFF, CAP ON  →  MAXIMAL expression

7. Key Mutations and Their Effects

Mutation	Effect
lacI⁻ (no repressor)	Constitutive expression - genes always ON
lacIˢ (super-repressor)	Repressor can't bind allolactose; genes always OFF
Operator mutation (O⁻ˢ)	Repressor can't bind operator; constitutive expression
cya⁻ (no adenylyl cyclase, no cAMP)	Reduced expression even with inducer; CAP can't be activated
crp⁻ (no CAP)	Reduced expression; positive regulation lost

8. Clinical/Biochemical Significance

The lac operon is the founding model for understanding gene regulation, operons, and transcription factor-DNA interactions
IPTG-inducible expression systems derived from this operon are used universally in recombinant protein production
Allolactose acting as inducer (not lactose itself) is a key concept: the cell must first detect and metabolize the sugar before amplifying its own import machinery

Sources: Harper's Illustrated Biochemistry, 32nd Ed., pp. 433-436; Basic Medical Biochemistry: A Clinical Approach, 6th Ed., pp. 507-509; Jawetz, Melnick & Adelberg's Medical Microbiology, 28th Ed.

This is a shared conversation. Sign in to Orris to start your own chat.