Paper : 15 Molecular Cell Biology

Module : 23 Regulation of gene expression in Prokaryotes: Lac Operon

Development Team
Principal Investigator : Prof. Neeta Sehgal
Department of Zoology, University of Delhi

Co-Principal Investigator : Prof. D.K. Singh

Department of Zoology, University of Delhi

Paper Coordinator : Prof. Kuldeep K. Sharma

Department of Zoology, University of Jammu

Content Writer : Dr. SudhirVerma

Deen Dayal Upadhyaya College, University of Delhi

Content Reviewer : Prof. Rup Lal

Department of Zoology, University of Delhi

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 Description of Module

Subject Name ZOOLOGY

Paper Name Zool 015: Molecular Cell Biology

Module Name/Title Regulation of gene expression in Prokaryotes

Module ID M23: Lac Operon

Keywords Operon, Inducible Operon, Repressible Operon, Catabolite repression,

β-galactosidase, Permease, Transacetylase, Operator, Promoter,
Regulator, Structural genes

Contents

1. Learning Outcomes
2. Introduction
3. Regulation of Gene expression in Prokaryotes
4. Types of Gene Expression: Constitutive, Inducible and Repressible
5. The Positive or Negative Control of Gene Expression
6. Concept of Operon
7. The Lac Operon of [Link]
8. Regulation of Lac Operon
9. Substitutes of allolactose, used for Lac Operon
10. Lac Mutations
10.1. Mutations in structural genes
10.2. Mutations in regulator gene
10.3. Mutations in operator
10.4. Mutations in promoter
11. Positive Control: Catabolite Repression
12. Summary

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 1. Learning Outcomes
After studying this module, you shall be able to

 Know: What is an Operon system in prokaryotes? What are the various types of Operon
that exist in Bacteria? How lactose metabolism take place in bacteria?

 Learn: About various mechanisms of gene expression regulation in prokaryotes and how
an Operon system contributes at transcriptional level gene regulation.

 Identify: The components that constitute an Operon and regulate the prokaryotic gene
expression

2. Introduction
The genetic composition of all the cells within an organism is same yet they do differ from
each other in terms of structure and function due to differential expression of genes which
leads to difference in their protein composition. How this differential expression (which
might be temporal or spatial) is achieved? The answer lies in the ‘mechanisms of
transcriptional regulation’, also called as gene regulation. We will understand the gene
regulation in prokaryotes with an example of lactose metabolizing enzymes. But before that,
we shall briefly learn about the gene expression regulatory checkpoints in prokaryotes, the
Operon concept and general regulatory principles of transcription in bacteria.

3. Regulation of Gene expression in Prokaryotes

The gene expression can be broadly regulated at five different levels in prokaryotes:

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 Fig. 1: Levels of gene regulation in prokaryotes.
Source: [Link]

For the current module, we will restrict our discussion to transcriptional regulation only.

4. Types of Gene Expression: Constitutive, Inducible and Repressible

Types of Gene Expression:

Gene expression can be:

 Constitutive Gene Expression

 Inducible Gene Expression
 Repressible Gene Expression

Constitutive Gene Expression refers to the genes which are constitutively expressed in
most cells e.g. genes which express housekeeping function proteins like ribosomal
proteins, tRNA, rRNA etc. There are other genes whose products are required for cell
growth only under certain environmental conditions only. Thus, constitutive expression of
such genes is a waste of energy. The expression of such genes is switched ON only when
their product is needed. This process of turning on the expression of genes in response to a
substance in the environment is called the Inducible gene expression and these genes are

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 called as inducible genes. The catabolic pathway enzymes such as in lactose, galactose etc.
are example of inducible expression. In contrast, the continued synthesis of some
biosynthetic enzyme is wastage if that molecule is present in the environment where
bacteria are growing. Thus, in case of externally available nutrient molecule, the enzymes
required for biosynthesis of that nutrient needs to be turned off. Such an expression is
called as repressible gene expression.

5. The Positive or Negative Control of Gene Expression

If the product of regulator gene is involved in turning on the expression of one or more
structural genes, the control mechanism is termed as Positive Control Mechanism. And, if
the product of regulator gene is involved in turning off the expression of structural genes,
the control mechanism is termed as Negative Control Mechanism.

Fig. 2: Different types of gene expression control mechanisms i.e. negative inducible, negative repressible,
positive inducible and positive repressible. (Source: [Link]/[Link])

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 6. Concept of Operon
The significant difference between bacterial and eukaryotic gene control lies in the
organization of functionally related genes. The bacterial genes which have related
functions are clustered and under the control of a single promoter. These genes are
transcribed together in a single mRNA. Thus, in prokaryotes, the genes with related
functions are coordinately regulated genetic units called the ‘Operons’. The Operon model
was developed by Francois Jacob and Jacques Monod in 1961 (For which they were
awarded Nobel Prize in physiology and medicine in year 1965) to explain the regulation of
genes required for utilization of lactose in [Link]. The organization of a typical Operon is
as follows:

Fig. 3: Diagrammatic representation of a typical Operon

Source: [Link]

Source: [Link]

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Regulation of gene expression in Prokaryotes: Lac Operon
 At one end of Operon is present a set of structural genes e.g. gene A, B and C. These genes
are coordinately expressed in a single mRNA which is further translated to form protein A,
B and C respectively. Upstream of these structural genes is present a promoter which
controls the transcription of all these structural genes. The RNA polymerase binds to this
promoter and then goes downstream to transcribe the structural genes. A regulator gene
also helps in controlling the transcription of structural genes. The regulator gene has its
own promoter and not actually a part of Operon. The regulatory protein can bind to a
region on Operon, called as Operator. The operator usually overlaps the 3’ end of promoter
and 5’ end of first structural gene. Some Operons may have multiple operators as well.

The Operons can be inducible Operon (in which transcription is normally OFF and
something must happen to switch it ON) and repressible Operon (in which transcription
is normally ON and something must happen to turn it OFF).

Fig. 4: Some common Operons in prokaryotes

Source: [Link]

7. The Lac Operon of E. coli

Francois Jacob and Jacques Monod explained the Operon model for genetic control of
lactose metabolism in E. coli in in 1961. Their work and the subsequent research
established ‘Operon’ as basic unit of transcriptional regulation in bacteria. Though no
sequencing methods were available to determine the nucleotide sequence at that time, but
Jacob and Monod deduced the Operon structure genetically by analyzing the interactions
in mutations that interfered the lactose metabolism regulation. We’ll discuss these

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 mutations in subsequent sections but before that let’s first understand how lactose
metabolism is regulated by lac Operon in bacteria.

Lactose metabolism in E. coli: The E. coli bacteria present in our gut helps in metabolizing
the sugar Lactose which is the major carbohydrate found in milk. The lactose is not
internalized passively by E. coli but actively, by a protein called permease. Once lactose is
transported inside the bacterial cell, it is broken up into its constituent monosaccharide i.e.
glucose and galactose by enzyme, β-galactosidase. The same enzyme can also convert the
lactose into allolactose also which is involved in regulation of lactose metabolism. Once
converted into monosaccharides, it can be used as energy source by bacteria. Yet another
enzymes which is produced by lac Operon is thiogalactoside transacetylase whose
function is not yet clearly understood.

Fig. 5: Catalytic action of β-galactosidase cleaving lactose into glucose and galactose. (Source: Author)

Fig. 6: Catalytic action of lac Operon genes in transport and metabolism of lactose in bacteria.
Source: Modified from Lehninger Principles of Biochemistry, 4th Ed. (2006)
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Regulation of gene expression in Prokaryotes: Lac Operon
 8. Regulation of Lac Operon
Lac Operon is an example of negative inducible Operon. In a negative inducible Operon,
an active repressor is encoded by a regulator gene which readily binds to the operator.
Binding of repressor protein to operator blocks the binding of RNA polymerase to
promoter as operator site overlaps with promoter. Thus, no transcription takes place. For
transcription to occur promoter should be free or indirectly, the repressor should not bind
to operator site. The transcription in such a case is normally inhibited (or in OFF state) and
needs to be induced (or turned ON). In order to switch on the transcription, an inducer
binds to repressor. The repressor proteins are usually allosteric in nature. They usually
have two binding sites: one for DNA and another for some other molecule such as inducer.
Thus, when inducer binds to repressor, it alters its shape which leads to dissociation of
repressor from DNA. Simple, presence of inducer switches ON the transcription leading to
expression of structural genes whereas absence of inducer keeps the transcription OFF
because of repressor leading to no expression of structural genes.

Structurally, lac Operon consists of a common promoter (lacP) and three adjacently
located structural genes: lacZ, lacY and lacAwhich encode for β-galactosidase, permease
and transacetylase respectively. Between the operator and promoter lies the operator
(lacO). Upstream of promoter is present the regulator gene lacI, which has its own
promoter (PI). lacI encodes for a repressor which has two binding sites: one for DNA and
another for allolactose. Allolactose acts as an inducer for lac Operon and is produced by
action of β-galactosidase on lactose.

Fig. 7: Structural arrangement of lac Operon in [Link]

Source:
[Link]
[Link]

ZOOLOGY Molecular Cell Biology

Regulation of gene expression in Prokaryotes: Lac Operon
 Fig. 8: Size of gene, gene products and their molecular weights involved in lac Operon
Source:
[Link]
&ved=0ahUKEwj96aC51PTKAhWPG44KHewuBngQ_AUIBygC#tbm=isch&q=lac+Operon+creative+commo
ns&imgrc=xi9tdINNkOq5_M%3A

Table 1: Function of structural genes of lactose Operon (Source: Author)

Structural genes of the Lac Operon

Gene Protein (Enzyme) Coded Function
lacZ β-galactosidase Catabolism of lactose to glucose and
galactose
lacy Permease Transport of lactose in the cell
lacA Transacetylase Largely unknown but probably it rids the cell
of toxic thiogalactosides.

When lactose is absent; no allolactose is formed and hence no inducer is present. The
repressor binds to operator and blocks the promoter site. The RNA polymerase is unable to
bind to promoter and thus, no transcription takes place causing no expression of structural
genes required for lactose metabolism. In fact, it makes the physiological sense. If no
lactose is present in the growth medium, there is no requirement to form the enzymes/
proteins to catabolise it and energy is saved.

When lactose is present; initially some of the lactose is converted to allolactose which then
binds to repressor and causes a structural change in it leading to its release from DNA.
Now, the promoter is free and RNA polymerase can bind to it and transcription of lac
Operon genes takes place. It also makes physiological sense to synthesize the lactose
metabolizing enzymes only when lactose is present in the growth medium to metabolize.

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Regulation of gene expression in Prokaryotes: Lac Operon
 In what we have discussed above, you must have realized that:

1. Permease is required to transport lactose into the cell. But, if lac Operon is repressed
how lactose does get into the cell to inactive the repression and turn on transcription?

Secondly,

2. The inducer for lac Operon is allolactose, which is formed from lactose. But, if β-
galactosidase production is repressed, how can lactose metabolism be induced?

The answer for these questions is that repression doesn’t completely shuts down the
transcription of lac Operon. There is always a low level of transcription, even if the
repressor is actively bound to operator. Thus, a few molecules of β-galactosidase,
transacetylase and permease are always present in the cell. When lactose appears in the
medium, permease transports it inside the cell and β-galactosidaseconverts it into
allolactose which then induces the transcription.

The lac Operon is ‘Leaky”.

9. Substitutes of allolactose, used for Lac Operon

Besides allolactose, many similar compounds are used as inducer for lac Operon.
Isopropylthiogalactoside (IPTG) is one such molecule which is extensively used in
research to observe the effects of induction, independent of metabolism as it’s not
metabolized by β-galactosidase.

Fig. 9: Structures of ‘inducers’ of Lac Operon (Source: Author)

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Regulation of gene expression in Prokaryotes: Lac Operon
 10. Lac Mutations
The structure and function of lac Operon was worked out by Jacob and Monod by
analyzing the mutations that affected lactose metabolism. They used partial diploid strains
of E. coli. These strains possess two different DNA molecules: one complete bacterial
chromosome and an extra piece of DNA. Jacob and Monod created such strains by
conjugation process. As we know, in conjugation, small circular piece of DNA i.e. F
plasmid is transferred from one bacterium to another. Jacob and Monod used the F plasmid
of lac Operon. So, the recipient bacterium became partially diploid as it possessed two
copies of lac Operon. They used different combination of mutations on F plasmid and
bacterial DNA and deduced that some of the lac Operon components are cis-acting
(controlling the gene expression only when present on same chromosome) and others are
trans-acting (controlling the gene expression on other DNA molecules). The experiments
in which genes or gene clusters are tested pairwise are called as complementation tests.

Let us discuss the mutations in all the components of lac Operon i.e. in structural gene,
regulator gene, operator and promoter.

10.1. Mutations in structural genes

Using partial diploids, Jacob and Monod established that mutations at lacZ and lacY genes
were independent and usually affected the product of the gene in which they occurred. The
partial diploid with lacZ+lacY- on bacterial chromosome and lacZ-lacY+ on plasmid
functioned normally, producing permease and β-galactosidase in presence of lactose. The
single functional β-galactosidase gene (lacZ+) in this partial diploid is sufficient to form β-
galactosidase irrespective of its coupling with defective (lacY-) or functional (lacY+)
permease. The same is applicable for lacY+ gene.

10.2. Mutations in regulator gene

Mutations in lacI gene affect both β-galactosidase and permease as both the genes are in
same Operon and regulated in co-ordinate manner. The mutations which were constitutive
in nature i.e. causing lac proteins to be produced all the times irrespective of whether
lactose was present or not were designated as lacI-. The lacI+ gene is dominant over lacI-
gene; means a single copy of lacI+ gene is sufficient to bring normal regulation. Further,
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Regulation of gene expression in Prokaryotes: Lac Operon
 lacI+ is trans-acting in nature; means it can regulate the Operon normally even if Operon is
located on some other DNA molecule.

Some lacI mutations did not allow transcription to take place even in the presence of
lactose and other inducers e.g. IPTG. Such mutations are referred as super repressor
mutations (lacIs). The repressor produced by such mutations is defective in a way that
inducer could not inactivate it. The lacIs mutations are dominant over lacI+. The partial
diploids with genotype lacIslacZ+/ lacI+lacZ+ were unable to synthesize permease and β-
galactosidase irrespective of presence or absence of lactose.

10.3. Mutations in operator

The mutations in operator region are referred as lacOc (denoting constitutive mutations on
operator region). These mutations alter the operator DNA sequence such that repressor is
unable to bind it. A partial diploid with genotype lacI+lacOclacZ+/ lacI+lacO+lacZ+ showed
constitutive expression of β-galactosidase indicating that lacOc is dominant over lacO+.
Additionally, lacO is cis-acting in nature; means it affects the genes present on same DNA
molecule only. The partial diploid with genotype lacI+lacO+lacZ-/ lacI+lacOclacZ+ was
constitutive in β-galactosidase production with or without lactose. But, a partial diploid
genotype lacI+ lacO+lacZ+/ lacI+lacOclacZ- produced β-galactosidase only in the presence
of lactose. In lacI+lacO+lacZ-/ lacI+lacOclacZ+ genotype, both lacOc and functional lacZ+
are present on same DNA but in lacI+ lacO+lacZ+/ lacI+lacOclacZ-, the lacOcand functional
lacZ+ are present on different DNA. The lacOmutations regulate genes present on same
DNA only by preventing binding of repressor to operator sequence but they cannot prevent
a repressor from binding to a normal operator sequence present on some other DNA.
Hence they are cis-acting.

10.4. Mutations in promoter

Mutations at promoter region are denoted as lacP-. These mutations interfere with binding
of RNA polymerase to promoter region hence no transcription. Thus, lacP- mutation leads
to no transcription of structural genes irrespective of whether lactose is present or not. Like
operator mutations (lacOc), promoter mutations (lacP-) are also cis-acting.

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Regulation of gene expression in Prokaryotes: Lac Operon
 Fig. 10: Lac regulatory mutants (lac A is omitted for simplicity). (a)- (d) show haploid states i.e. cell carries a
single copy of lac genes. (a) shows repression, (b) shows IPTG mediated induction, (c) and (d) show effect of
mutation to lacI gene or operator respectively.(e) shows complementation test for repressor is shown. (f) and (g)
show complementation test for operator mutation which is cis-dominant.
Source:[Link]

11. Positive Control: Catabolite Repression

Glucose is always the preferred source of energy over other sugars (such as, lactose,
arabinose, maltose etc.) and thus, is always the first one to be metabolized in presence of
other sugars. If E. coli is grown in a medium which has both glucose and lactose, it always
prefers to consume glucose first and then the lactose. Hence in such a growth medium, E.
coli shows diauxic growth as depicted in biphasic growth curve below. During the first
phase (I) of exponential growth the bacteria will utilize glucose as source of energy until
all the glucose is exhausted. Then it will utilize lactose and so, after a secondary lag phase
it will show a second phase (II) of exponential growth.

Fig. 11: Biphasic growth curve of [Link], grown in presence of glucose and lactose
Source: [Link]
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Regulation of gene expression in Prokaryotes: Lac Operon
 The lac promoter contains two binding sites: one for RNA polymerase and another one for
protein, called as ‘catabolite activator protein (CAP)’. The binding of CAP to promoter
prevents the Operon from being induced in presence of glucose. Simply, this mechanism
ensures the preferential utilization of glucose as energy source if its available in growth
medium, which makes the physiological sense to avoid waste of energy in making lactose
catabolising enzymes when ready source of energy i.e. glucose is available. This
phenomenon is called as ‘Catabolite Repression”.

If the glucose is present in growth medium, it prevents the induction of lac Operon.
Glucose, which is a catabolite of lactose metabolism is a repressor of lac Operon, hence
this phenomenon is termed as “Catabolite Repression’ (also called as glucose effect or
positive regulation). The regulatory protein CAP and cAMP molecules mediate this type of
regulation. CAP is also called as CRP i.e. cAMP receptor protein because CAP binds to
cAMP when cAMP is present in sufficient concentrations. The Cap/cAMP complex must
be present at its binding site on lac promoter in order to lac Operon being induced
normally. Since it exerts a positive control on lac Operon, it is called as positive regulator
and phenomenon is called as positive control of lac Operon induction. The CAP/ cAMP
complex bends the DNA by 90° when it is bound to it. Since CAP/ cAMP complex
binding site and RNA polymerase binding sites are located adjacently, this bending makes
the RNA polymerase binding site more accessible and thus positively regulate the
induction of lac Operon.

Fig. 12: Structure of CAP protein and its binding sequence. (A) Two identical CAP dimer subunits bind to the
activator binding sequence and induce a bend in the DNA (by ~80°). A molecule of cAMP (coactivator) is

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Regulation of gene expression in Prokaryotes: Lac Operon
 bound to each monomer. (B) Symmetric half sites of the CAP binding sequence possessing dyad axis. CAP
binding site is centered at position −61.5.
Source:[Link]
binding_domain[Link]
5._Positive_and_negative_control_of_gene_expression

It is the CAP/ cAMP complex that binds to lac promoter and not the CAP alone which means
cAMP is major effector molecule. The intracellular concentration of cAMP is affected by
presence or absence of glucose. Low concentration of glucose causes higher concentration of
cAMP and vice-versa showing an inverse relationship. The glucose prevents the activation of
adenylatecycalse, the enzyme which catalyzes the formation of cAMP from ATP. The
connection between glucose and cAMP involves the mechanisms by which sugars such as
glucose are transported into the bacterial cell and fact that adenylatecyclise is a membrane
bound enzyme.

Many sugars are transported in E. coli by phosphoenolpyruvate-dependent

phosphotransferase systems (PTS). They are phosphorylated as soon as they are
transported into the cells. The phosphate group is transferred from phosphoenol pyruvate
(PEP) through a series of intermediary proteins. The pathway is summarized below:

(A)

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Regulation of gene expression in Prokaryotes: Lac Operon
 (B)

Fig. 13: A) The link between adenylatecyclase activation and glucose transport.
B) The phosphoenolpyruvate-dependent phosphotransferase system (PTS)
Source: Author

The connection between sugar transport and cAMP involves the interaction of EIIA~P and
adenylatecyclase. EIIA~P activates adenylatecyclase. Thus,

If Glucose is scarce, the EIIA~P will be plentiful, adenylatecyclase will be activated and
cAMP will increase and vice-versa.

TABLE 2: Signals that Induce or Repress Transcription of the Lac Operon

Source: Author
Glucose CAP binds Lactose Repressor binds Transcription
+ - - + No
+ - + - Some
- + - + No
- + + - Yes

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Regulation of gene expression in Prokaryotes: Lac Operon
 Also, when glucose is absent, the EIIA will be less and thus, lactose permease transport will
not be inhibited by EIIA.

12. Summary

 Though the genetic composition of all the cells within an organism is same, the
difference in cells in terms of structure and function exists. This is because of
differential expression of genes which leads to difference in their protein
composition.

 This differential expression (which might be temporal or spatial) is achieved by

‘mechanisms of transcriptional regulation’, also called as gene regulation.

 The gene expression can be broadly regulated at five different levels in prokaryotes
i.e. at transcription level, RNA processing level, RNA transport level, Translational
level and Post-translational modification level.

 Gene regulation at transcription level in prokaryotes can be constitutive, inducible

or repressible.

 It can be positive regulation as well as negative regulation.

 The significant difference between bacterial and eukaryotic gene control lies in the
organization of functionally related genes. The bacterial genes which have related
functions are clustered and under the control of a single promoter. These genes are
transcribed together in a single mRNA. Thus, in prokaryotes, the genes with related
functions are co-ordinately regulated genetic units called the ‘Operons’.

 The Operon model was developed by Francois Jacob and Jacques Monod in 1961
to explain the regulation of genes required for utilization of lactose in [Link].

 An Operon consists of some structural genes, operator, promoter and regulator

genes.

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Regulation of gene expression in Prokaryotes: Lac Operon
  In lactose Operon, the structural genes are β-galactosidase, permease and
transacetylase. These genes synthesize the lactose metabolizing enzymes, if lactose
is present in the growth medium.

 The lac Operon is tightly regulated in a way that these genes are transcribed only
when lactose is present otherwise transcription remains largely OFF. However,
some transcription is also on, making lac Operon ‘leaky’.

 Lac Operon is a negative inducible Operon.

 Besides allolactose, IPTG also acts as substitute for lac induction.

 The structure and function of lac Operon was worked out by Jacob and Monod by
analyzing the mutations that affected lactose metabolism. They used partial diploid
strains of [Link]. The experiments in which genes or gene clusters are tested pairwise
are called as complementation tests.

 [Link] shows diauxic growth if grown in medium consisting both glucose and lactose
as glucose is preferential source of energy

 Glucose acts as catabolite repressor of lac Operon.

 Lac Operon is positively regulated by CAP-cAMP complex.

 There is a connection between glucose transport and cAMP formation as both the
mechanism share common system i.e. phosphoenolpyruvate-dependent
phosphotransferase systems (PTS).

 Thus, lac Operon is an example of negative inducible Operon in prokaryotes which is

also regulated by CAP-cAMP complex and repressed by presence of glucose in the
medium.

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Regulation of gene expression in Prokaryotes: Lac Operon
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Regulation of gene expression in Prokaryotes: Lac Operon