The lac operon
Lactose catabolism
• In bacteria, the genes involved in the
same process are often clustered together.
For example, the genes that allow E. coli
to break down milk sugar (lactose) to
produce energy.
Lactose catabolism
• lacY encodes lactose permease transports lactose into the cell
• lacZ encodes -galactosidase –
enzyme that catalyses the reaction:
lactose  glucose + galactose
• lacA encodes lactose transacetylase –
biological function unclear.
Lactose catabolism
• These genes are controlled. E. coli is a
successful competitor in the gut because it
doesn’t waste time and energy making
mRNA and proteins that are not needed. The
lac genes are only transcribed if lactose is
present in the growth medium.
• These genes are expressed co-ordinately.
Either they are all switched on or they are all
switched off.
Lactose catabolism
• The coordinate regulation arises from the
clustering of the genes (strictly called
CISTRONS) into a structure called an OPERON.
• There is also a regulatory gene, the lacI gene, that
is not part of the operon. This produces a
repressor protein that controls the operon.
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Transcription
mRNA
LacI
repressor
lactose
Active
repressor binds
to operator
Inactive repressoreffector complex
(polycistronic message)
translation
-galactosidase
enzyme
protein
lactose
permease
lactose
transacetylase
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Transcription
mRNA
(polycistronic message)
translation lie side
three Active
coding sequences
The repressor binds
by
LacI
to operator
protein
side
but
there
is
only
one
promoter
repressor
lactose
Inactive repressoreffector complex
-galactosidase
enzyme
lactose
permease
lactose
transacetylase
The lac operon
This means
that
there
is
only
one
mRNA
that
encodes
P lacI
P lacO
lacZ
lacY lacA
three proteins. Each coding region has its own start and
DNA
stop
codon
RNA
Transcription
polymerase
mRNA
LacI
repressor
lactose
Active
repressor binds
to operator
Inactive repressoreffector complex
(polycistronic message)
translation
-galactosidase
enzyme
protein
lactose
permease
lactose
transacetylase
P lacI
P lacO
RNA
polymerase
LacI
repressor
lactose
operon
The separateThe
lacIlac
gene
is
not controlled. It has
lacZ
lacY lacA
Its own promoter and
encodes a repressor DNA
protein. Transcription
It is not part of
the operon
mRNA
Active
repressor binds
to operator
Inactive repressoreffector complex
(polycistronic message)
translation
-galactosidase
enzyme
protein
lactose
permease
lactose
transacetylase
P lacI
RNA
polymerase
LacI
repressor
lactose
InThe
the absence
of
lac operon
lactose, the repressor
protein
P lacO
lacZ
lacY binds
lacA to a
special site in the
DNA
operon called the
Transcription
OPERATOR and
mRNA
prevents RNA
(polycistronic message)
polymerase from
Active
translation
repressor binds
moving along the
to operator
protein
DNA
Inactive repressoreffector complex
-galactosidase
enzyme
lactose
permease
lactose
transacetylase
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Transcription
mRNA
LacI
repressor
lactose
Active
repressor binds
to operator
Inactive repressoreffector complex
(polycistronic message)
translation
-galactosidase
enzyme
protein
lactose
permease
lactose
transacetylase
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Lactose absent:
operon switched off
mRNA
LacI
repressor
Active
repressor binds
to operator
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Transcription
mRNA
(polycistronic message)
translation
LacI
repressor
lactose
protein
Inactive repressoreffector complex
-galactosidase
enzyme
lactose
permease
lactose
transacetylase
The lac operon
Jacob and Monod
Jacob
François Jacob and
Jacques Monod worked
out how the lac operon
functioned and they
formulated the operon
hypothesis.
Monod
The lac operon
lac mutants
The properties of various mutants
allowed Jacob and Monod to work
out how operons work.
The lac operon
P O
lacZ
lacY
lacA
DNA
mRNA
Active -galactosidase
enzyme
lacZ mutations
are recessive
protein
Lactose catabolism
X
DNA
X
X
mRNA
protein
The lac operon
Constitutive mutants
• Mutants where the lacI gene has
mutated, will grow on lactose.
• However they make β-galactosidase
all of the time. These mutants that
have lost the ability to control gene
expression are called constitutive
mutants. They are also recessive.
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Active repressor binds
to both operators
LacI
repressor
P lacI
P lacO
lacZ
lacY
lacA
X
No active
repressor to X
bind to
operator
lacI mutations
are recessive
The lac operon
A testable prediction
• Jacob & Monod realised that if their operon
hypothesis was right, there should be another type
of constitutive mutant – one where the operator has
mutated so that the repressor cannot recognise it.
• Such mutants should be dominant and it should be
possible to isolate them in a diploid.
The lac operon
P lacI
P lacO
lacZ
lacY
lacA
DNA
RNA
polymerase
Active repressor binds only
to wild type operator
LacI
repressor
P lacI
P lacO
lacZ
lacY
lacA
X
DNA
mRNA
lacOc mutations are dominant
The lac operon
• Jacob & Monod mutated a diploid wild
type to see whether they could get
constitutive mutants.
• They did get them, and showed that they
mapped to the operator region.
This supported their hypothesis.
The lac operon
The lac repressor is an example of a
negative regulatory protein, whose action
prevents expression of the genes under its
control and whose function is controlled by
an effector molecule (in this case, lactose).
The lac operon
Catabolic repression
The lac operon is also under the control of a positive
regulatory protein.
E. coli’s preferred carbon source is glucose.
Glucose inhibits transcription of the lac operon, even
in the presence of lactose.
Inhibition occurs in lacI and lacO mutants, as well as
wild type, indicating the effect of glucose is NOT via
the repressor-operator interaction.
The lac operon
The effect of glucose is mediated by a nucleotide,
cyclic AMP (cAMP).
The intracellular concentration of cAMP is high in the
absence of glucose and low in its presence.
cAMP binds to a catabolic activator protein (CAP),
upstream of the lac promoter driving the lac operon.
When bound to cAMP, CAP enhances lac
transcription.
The lac operon
P lacO
lacZ
lacY
lacA
CAP
DNA
RNA
polymerase
Transcription
mRNA
(polycistronic message)
translation
protein
Glucose
cAMP
-galactosidase
enzyme
lactose
permease
lactose
transacetylase
The lac operon
Summary
Regulation of expression of the lac operon is under
two sets of controls, both of which are governed by
environmental factors.

The repressor-operator interaction provides an
“all or none” level of control (lactose  on).

[CAP-cAMP]-CAP-binding site interaction
provides a modulatory control.
(glucose levels control rate of mRNA initiation)
Complementation
• Diploid, Haploid
• Dominant, Recessive
• Homozygous, Heterozygous
• Cistron
• Cross-feeding
Colinearity of the gene and protein
 Protein structure
 Haemoglobin
 Genetic code  amino acid sequence
The Genetic code




Codon
Dictionary of the genetic code
How the code was deciphered
How the code works
tRNA and Translation






RNA translation (5'  3')
Ribosomes
Structure of tRNA
Anticodon
Mechanism of translation
Wobble hypothesis
Inosine (I) is a
rare base
found in
tRNA, often in
the anticodon,
capable of
binding to
adenine, uracil
or cytosine.
RNA Translation





RNA growth (5'  3')
RNA polymerase, structure and properties
Promoter, consensus
Mechanism of translation
Termination
Suggested reading
Regulation of gene transcription (2000) In: An
Introduction to Genetic Analysis. pp 336-344.
Griffiths, A. J. F,. Miller, J. H., Suzuki, D. T.,
Lewontin, R. C. and Gelbart, W. M. (Eds). Freeman
and Company, New York.