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Lecture # 13 (Gene Regulation in Prokaryotes - Lac Operon)

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38 views25 pages

Lecture # 13 (Gene Regulation in Prokaryotes - Lac Operon)

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Aar kyy
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We take content rights seriously. If you suspect this is your content, claim it here.
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Gene Regulation

“Lac operon”
Introduction
• What causes gene products to be synthesized in some
cells under some conditions, but not in others? A large
part of research in molecular biology in aimed at trying to
determine this. We are going to talk about a few basic
systems, but a whole course could easily be devoted to
the subject.
• For most genes, the essential regulation point is
transcription: whether the gene is transcribed or not.
Regulation also occurs at other points: availability of the
DNA to be transcribed at all, whether the mRNA is
translated, stability of the mRNA, how quickly the protein
is degraded, etc.
Gene Regulation in Prokaryotes
• The first system of gene regulation that was
understood was the lac operon in E. coli, worked
out by Francois Jacob and Jacques Monod in
1962. Many other prokaryotic genes are
regulated in a similar fashion, and the basic
principles carry over into eukaryotes.
• The lac operon codes for enzymes involved in
the degradation of lactose. Lactose is a
disaccharide that can be used as food in the
absence of glucose. A lac- mutant is a
chemoauxotroph that can’t use lactose.
Operons

• An operon is a group
of genes that are
transcribed at the
same time.
• They usually control
an important Jacob, Monod & Lwoff
biochemical process. © NoblPrize.org

• They are only found


in prokaryotes.
Operon
• Promoter (RNA Polymerase is bound)
• Operator (Repressor is bound)
• Several Structural Genes
• Terminator

repressor – regulatory gene, allosteric protein


corepressor – product molecule
inducer – substrate molecule
Inducible versus repressible operons

• a. Inducible operons are turned on in reponse to a


metabolite (a small molecule undergoing metabolism)
that regulates the operon. E.g. the lacoperon is induced
in the presence of lactose (through the action of a
metabolic by-product allolactose).

• b. Repressible operons are switched off in response to


a small regulatory molecule. E.g., the trp operon is
repressed in the presence of tryptophan.
Adapting to the environment

• E. coli can use either glucose, which is a


monosaccharide, or lactose, which is a
disaccharide.
• However, lactose needs to be hydrolysed
(digested) first.
• So the bacterium prefers to use glucose
when it can.
Four situations are possible
1. When glucose is present and lactose is absent the E.
coli does not produce β-galactosidase.

2. When glucose is present and lactose is present the E.


coli does not produce β-galactosidase.

3. When glucose is absent and lactose is absent the E.


coli does not produce β-galactosidase.

4. When glucose is absent and lactose is present the E.


coli does produce β-galactosidase.
Structure of the lac Operon
• The lac operon consists of 3 protein-coding genes plus
associated control regions.
• The 3 genes are called z, y, and a. lacZ codes for the
enzyme beta-galactosidase, which splits lactose into
glucose plus galactose. lacY codes for a “permease”
protein that allows lactose to enter the cell, and lacA
codes for an enzyme that acetylates lactose. Together
these three genes are called the “structural genes”. We
will mainly focus on lacZ.
• All 3 genes of the lac operon are transcribed on the
same messenger RNA. Ribosomes translate the 3
proteins independently. This is a feature of prokaryotes
that is only very rarely seen in eukaryotes, where 1 gene
per mRNA is the rule.
Control Regions
• Near the lac operon is another gene, called lacI, or just
“i”. It codes for the lac repressor protein, which plays an
essential role in lac operon control. The lac repressor
gene is expressed “constitutively”, meaning that it is
always on (but at a low level). It is a completely separate
gene, producing a different mRNA than the lac operon.
• Just upstream from the transcription start point in the lac
operon are two regions called the operator (o) and the
promoter (p). Neither region codes for protein: they act
as binding sites on the DNA for important proteins.
• The promoter is the site where RNA polymerase binds to
start transcription. Promoters are found upstream from
all protein-coding genes.
• The operator is where the actual control occurs.
Visual
Control
• The lac repressor protein (made by lacI) has 2 states: it can either
bind to lactose (technically, to a lactose derivative called allolactose)
or it can bind to the operator region of the lac operon.
• In the presence of lactose, the repressor binds to it, and the
repressor-lactose complexes float freely in the cytoplasm away from
the DNA. In this situation, RNA polymerase can bind to the
promoter, and the gene is transcribed. It makes beta-galactosidase
which digests the lactose.
• In the absence of lactose, the repressor binds to the operator DNA.
The repressor is a large molecule, and when it is bound to the
operator, RNA polymerase is blocked from reaching the promoter.
The lac operon is not transcribed, and no beta-galactosidase is
made.
• If lactose appears, the operon is said to be “induced”. The lactose
binds to the repressor, which then falls off the operator and allows
transcription to occur.
Lac Operon - Negative Regulation
Negative and Positive Regulation
• As described above, the lac operon is negatively
regulated: the regulatory protein (repressor)
causes transcription to stop.
• Positive regulation, where the regulatory protein
causes transcription to start, is more common.
• The lac operon also contains an example of
positive regulation, called “catabolite
repression”. E. coli would prefer to use glucose
as its food source. In the presence of glucose,
the lac operon (and other similar genes) are
turned off, even if lactose is present in the
medium.
Binding of cAMP-CAP to its site will enhance efficiency of
transcription initiation at promoter

• a. The lac promoter is not a particularly strong


promoter. The sequence at -10, TATGTT, does not
match the consensus (TATAAT) at two positions.
•
• b. In the presence of cAMP-CAP, the RNA polymerase
will initiate transcription more efficiently.
Regulatory region of lac operon, including
CAP binding site
Lac operon - positive regulation

Summary: Lac
operon is active only
in time, when the
activator CAP+cAMP
is attached onto
promotor, but when
is not present
represor onto
operator.
Catabolite Repression
• Catabolite repression uses a regulatory protein called CAP
(catabolite activator protein). It also uses the small molecule
cyclic AMP (cAMP).
• cAMP is made from ATP. When the glucose level in the cell is
high, the cAMP level is low, because glucose inhibits
synthesis of cAMP. When the glucose level is low, the cAMP
level is high.
• cAMP combines with the CAP protein to form a complex that
binds to part of the lac operon promoter. This complex bends
the DNA in a way that makes it much easier for RNA
polymerase to bind to the promoter. This allows transcription
to occur, but only if the lac repressor isn’t present.
• Thus, low glucose levels cause high cAMP levels. When
cAMP is high, it combines with CAP The CAP-cAMP complex
then binds to the promoter to allow transcription to occur.
• This is positive regulation because the binding of CAP to the
DNA causes transcription to occur.
Enhancers and Silencers
• Elements that are not a part of the promoter but
can either enhance (enhancer) or inhibit
(silencer) transcription at a manner that is
position- and orientation-independent.

1)An enhancers binds transcription factors


(activators) to activate transcription.

2)An enhancer can localize upstream, within, or


downstream of a gene.
Enhancers can occur in a variety of
positions with respect to genes
Enhancer Enhancer P Transcription unit

Upstream

Adjacent

Downstream

Internal Ex1 Ex2

Distal
Lac Operon
Link to study lac operon

• https://bio.libretexts.org/
Under_Construction/
Cell_and_Molecular_Biology_(Bergtrom)/
12%3A_Regulation_of_Transcription_and
_Epigenetic_Inheritance/
12.02%3A_Gene_Regulation_in__the_Lac
tose_(lac)_OperonProkaryotes-

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