GENE REGULATION

Unit objectives

• After studying this unit, you should be able to:

• 1. Know the reason why prokaryotes should regulate the
amount of proteins formed by a cell.
• 2. Define the word operon and name some operon systems
that exist.
• 3. Explain the lac operon and state why it is named that
way.
• 4. Draw and describe the structure of the lac operon.
• 5. Explain the structure of the lac operon in the presence
and absence of glucose.
• 6. Explain the concepts of positive feedback and negative
feedback with reference to the lac operon.
• Bacterial cells need to control protein
production so that there is no wastage of
energy, protein or enzymes that are not
required by the cell.
• Bacterial cells achieve this in three different
ways;
– rapid degradation of mRNA soon after translation.
– by allosteric interaction especially with enzymes.
– an operon.
 The operon system
• An operon is a cluster of functionally-related
genes that are controlled by a shared
operator.
• An operon system consists of regulatory
elements and structural genes.
• Operons consist of multiple genes grouped
together with a promoter and an operator.
• Operons are present in prokaryotes (bacteria
and archaea), but are absent in eukaryotes.
• In some situations multiple operons are
controlled by the same regulatory protein.
• Operons were first identified as a mode of
gene expression control in 1961 by François
Jacob and Jacques Monod.
 Operon Structure

• Operons are regions of DNA that contain

clusters of related genes.
• They are made up of a promoter region, an
operator, and multiple related genes.
• The operator can be located either within the
promoter or between the promoter and the
genes.
• RNA polymerase initiates transcription by
binding to the promoter region.
• The location of the operator is important as its
regulation either allows or prevents
transcription of the genes into mRNA.
Promotor, operator and regulator regions

• The promoter region codes for the Lac-P gene.

• The promoter region lies between the
regulator and the operator.
• RNA-polymerase binds to this site, as a
promoter is a region for initiation of
transcription.
• The promoter region is 100 base pairs long.
• It consists of palindromic sequences.
• This site promotes and controls the
transcription of structural genes or mRNA.
• The promoter site is regulated by the
regulatory genes of the repressor.
• The operator region codes the Lac-O gene.
• It lies between a promoter and the structural
gene (Lac-Z).
• It contains an operator switch, which decides
whether transcription should take place or
not.
• The operator region is where the regulatory
gene binds.
• The regulator region codes for the regulator
gene (Lac-I) that controls the activity of
promotor and an operator gene.
• This regulatory gene produces regulatory
proteins known as “Repressor proteins” which
can bind to the promoter and operator.
• The structural genes of the lac operon include
lac-Z, lac-Y and Lac-A.
• Lac-Z encodes for the enzyme beta
galactosidase which brings about the
hydrolysis of lactose into galactose and
glucose subunits.
• Lac-Y encodes for the enzyme lactose
permease (Galactoside permease).
• Lactose permease brings lactose into the cell.
• Lac-A encodes for the enzyme thiogalactoside
transacetylase (GAT).
• GAT transfers an acetyl group from acetyl-CoA
to galactosides, glucosides and lactosides.
• GAT is coded for by the lacA gene of the lac
operon in E. coli.
STRUCTURE OF AN OPERON
 Lac Operon

• The Lac operon is the classic operon example,

and is responsible for the degradation of the
milk protein lactose.
• The Lac operon is an inducible operon; in the
absence of lactose the operator is blocked by
a repressor protein.
• The operon is made up of a promoter with
operator, and three genes (lacZ, lacY, and lacA)
which encode β-galactosidase, permease, and
transacetylase.
• The three genes are involved in the
breakdown of lactose into its metabolites: β-
galactosidase breaks lactose down into
glucose and galactose, while the other two
proteins aid in the metabolic process.
• The expression of the Lac operon is controlled
by the regulatory gene lacI, located
immediately adjacent to the promoter region.
LacI encodes an allosteric repressor protein
that keep the Lac operon “off”.
• The lac operon is a bio-synthesis system that is
required for the efficient transport and
metabolism of lactose (milk sugar) in
Escherichia Coli and other related bacteria.
• In the absence of glucose, the E. coli bacteria
cells can utilise lactose as a source of energy,
however, it has to be processed via the lac
operon.
• When there is no lactose, the lac repressor
binds tightly to the operator.
• It gets in RNA Polymerase’s way, thereby
preventing transcription.
• In the presence of lactose ‘allolactose’
(rearranged lactose) binds to the lac repressor
and makes it let go of the operator.
• RNA polymerase can now transcribe the
operon.
• The same sequence of nucleotide bases which
constitute an operon also carry non-structural
genes that do not code for protein but aid the
operon in regulating the metabolism of
lactose.
• These extra genes on the operon are
collectively called regulator sites.
• Specifically, the lac operon consists of a
promoter site, a terminator site and an
operator site.
• Specific control of the lac genes depends on
the presence or absence of lactose in a cell.
• When lactose is absent in the bacteria growth,
the lac gene remain dormant because the
need to produce enzymes required to process
the lactose does not arise.
• When lactose is present enzyme β-galactoside
permease gets inserted into the membrane,
this allows lactose to flood into the cell at a
faster rate.
• Once the lactose is inside the cell, enzyme β-
galactosidase breaks down lactose into
simpler sugars: glucose and galactose.
• These simple sugars are then used as source
of energy by bacterial cells.
• The enzyme β-galactoside transacetylase
facilitates the transfer of acetyl group from
acetyl-CoA to β-galactoside.
• In this situation, the lac repressor protein
finally rolls-out.
• Since there is no more interference along the
lac operon, RNA polymerase can attach to the
promoter site and transcribe the lac operon
genes on to the mRNA.
• This happens as RNA polymerase moves along
the DNA from 3’ to 5’.
• As the lactose substrate runs out in the
medium, its absence induces a repressor
protein to go back to the original shape and
bind to the DNA operator site once again.
Operation of the Lac operon
Control of gene expression in prokaryotes

• Gene expression in prokaryotes is controlled

in two ways: positive control and negative
control.
• The positive inducible system (positive
control) is also known as ‘switch on’ of the lac-
operon.
• The positive control of gene expression in
prokaryotes occurs in the presence of lactose,
which is the inducer.
 Steps in positive control
• 1.The regulatory gene is expressed by the
repressor.
• 2.After expression of a regulatory gene, the
repressor produces repressor proteins.
• 3.Repressor protein has binding sites for the
operator and the inducer i.e. lactose.
• When lactose is present (inducer) it binds with
the repressor protein and forms “R+I
complex”.
• After the binding of the inducer to the
repressor, it blocks the binding of the
repressor to the operator.
• As the repressor protein does not block the
operator, the RNA polymerase binds to the
promotor and moves further to transcribe
mRNA.
 Negative control
• The negative control of the lac-operon is also
called the ‘switch-off’ mechanism.
• It occurs in the absence of the inducer
(lactose).
• Steps in negative control
• 1.First, the regulatory gene is expressed by the
repressor.
• After expression of a regulatory gene, the
repressor produces a repressor protein.
• In the absence of inducer or lactose the,
repressor protein directly binds to an
operator.
• This blocks the movement of RNA polymerase
and its attachment to the promoter.
• At last, inhibits the mRNA transcription.
Inducers and induction of lac-operon

• The inducer suppresses the activity and

binding of the repressor protein to the
operator and makes it “inactive repressor”
from the active repressor.
• In the Lac-operon, lactose or allolactose acts
as an inducer.
• Another inducer of the lac operon is
isopropylthiogalactoside (IPTG).
• Allolactose is formed by the enzyme beta-
galactosidase as a result of isomerization of
lactose (i.e. galactose links to the Carbon
6 instead of Carbon 4).
• In the absence of an inducer such as
allolactose or IPTG, the Lac I gene is
transcribed.
• The resulting repressor protein binds to the
operator site of the lac operon, Lac O, and
prevents transcription of the lac-Z, lac-Y and
lac-A genes.
• In the presence of inducers, the inducer binds
to the repressor.
• This causes a change in the conformation of
the repressor that greatly reduces its affinity
for the lac operator site.
• The lac repressor now dissociates from the
operator site and allows the RNA polymerase
(already in place on the adjacent promoter
site) to begin transcribing the lac-Z, lac-Y and
lac-A genes.
• These genes are transcribed to yield a single
polycistronic mRNA that is then translated to
produce all three enzymes in large amounts.
• The existence of a polycistronic mRNA ensures
that the amounts of all three gene products
are regulated co-ordinatedly.
• If the inducer is removed, the lac repressor
rapidly binds to the lac operator site and
transcription is inhibited almost immediately.
• High-level transcription of the lac operon
requires the presence of a specific activator
protein called catabolite activator protein
(CAP), also called cAMP receptor protein
(CRP).
• This protein, which is a dimer, cannot bind to
DNA unless it is complexed with 3’5′ cyclic
AMP (cAMP).
• The CRP–cAMP complex binds to the lac
promoter just upstream from the binding site
for RNA polymerase.
• It increases the binding of RNA polymerase
and so stimulates transcription of the lac
operon.
• Whether or not the CRP protein is able to bind
to the lac promoter depends on the carbon
source available to the bacterium.
• When glucose is present, E. coli does not need
to use lactose as a carbon source and so the
lac operon does not need to be active.
• The system has evolved to be responsive to
glucose.
• Glucose inhibits adenylate cyclase, the
enzyme that synthesizes cAMP from ATP.
• Thus, in the presence of glucose the
intracellular level of cAMP falls, so CRP cannot
bind to the lac promoter, and the lac operon is
only weakly active (even in the presence of
lactose).
• When glucose is absent, adenylate cyclase is
not inhibited, the level of intracellular cAMP
rises and binds to CRP.
• Therefore, when glucose is absent but lactose
is present, the CRP–cAMP complex stimulates
transcription of the lac operon and allows the
lactose to be used as an alternative carbon
source.
• In the absence of lactose, the lac repressor, of
course, ensures that the lac operon remains
inactive.
• These combined controls ensure that the lac-
Z, lac-Y and lac-A genes are transcribed
strongly only if glucose is absent and lactose is
present.
• The regulation that the lac operon undergoes
is termed negative inducible because gene is
turned off by the regulator factor (lac
repressor).
• The regulator gene produces a repressor
molecule which in the absence of lactose,
inhibits the structural genes directly but acts
through the operator gene.
• In eukaryotes gene regulation may occur
when DNA is uncoiled & loosened from the
nucleosomes (structural unit of a eukaryotic
chromosome consisting of a length of DNA
coiled around a core of histones) to bind to
transcriptional factors (epigenetic level), when
the RNA is transcribed (transcriptional level).
• When RNA is processed and exported to the
cytoplasm after it is transcribed (post-
transcriptional level), when RNA is translated into
protein (translational level), or after the protein
has been made (post-translational level). In
prokaryotes, RNA transcription and protein
translation occur simultaneously.
• Gene expression in prokaryotes is regulated at
the transcriptional level.
• Tryptophan operon (trp operon) in E. coli
Tryp operon encodes enzymes needed for the
biosynthesis of the amino acid tryptophan.
• This operon was discovered in 1953 by
Jacques monod.
• This operon has five structural genes that
code for the three enzymes required to
convert chorismic acid into tryptophan.