GENE REGULATION IN PROKARYOTES AND EUKARYOTES

GENE REGULATION
Sanju kaladharan

Prokaryotic Gene Regulation
Coordinate regulation of genesCoordinate regulation of genes
involved in similar functionsinvolved in similar functions

Types of Control
Negative ControlNegative Control Product of regulatory geneProduct of regulatory gene
inhibits transcriptioninhibits transcription
Positive ControlPositive Control Product of regulatory geneProduct of regulatory gene
enhances transcriptionenhances transcription

Operon
• Unit of coordinate gene expression
• Includes structural genes and their adjacent
regulatory elements
• We will consider
– Lac operon (inducible)
– Ara operon (inducible)
– Trp operon (repressible)

Types of Operons
InducibleInducible Initial condition: OFFInitial condition: OFF
Inducer switches operon ONInducer switches operon ON
RepressibleRepressible Initial condition: ONInitial condition: ON
Repressor switches operon OFFRepressor switches operon OFF

THE lac OPERON
© 2016 Paul Billiet ODWS

The control of gene expression
• Each cell in the human contains all the genetic
material for the growth and development of a
human
• Some of these genes will be need to be expressed all
the time
• These are the genes that are involved in of vital
biochemical processes such as respiration
• Other genes are not expressed all the time
• They are switched on an off at need.

Operons
• An operon is a group of
genes that are transcribed
at the same time.
• They usually control an
important biochemical
process.
• They are only found in
prokaryotes. Jacob, Monod & Lwoff

The lac Operon
• The lac operon consists of three genes each involved
in processing the sugar lactose
• One of them is the gene for the enzyme β-
galactosidase
• This enzyme hydrolyses lactose into glucose and
galactose.

Introduction
• regulator gene – A gene that codes for a
product (typically protein) that controls the
expression of other genes (usually at the level
of transcription).
• structural gene – A gene that codes for any
RNA or protein product other than a
regulator. Figure 26.01: A regulator gene
codes for a protein that acts at
a target site on DNA.

• In negative regulation, a repressor protein binds to an
operator to prevent a gene from being expressed.
• In positive regulation, a transcription factor is required
to bind at the promoter in order to enable RNA
polymerase to initiate transcription.
Figure 26.02: In negative control, a trans-
acting repressor binds to the cis-acting
operator to turn off transcription.
Figure 26.03: In positive control, a trans-
acting factor must bind to cis-acting site in
order for RNA polymerase to initiate
transcription at the promoter.

• The lac operon contains three genes: lacZ, lacY,
and lacA. These genes are transcribed as a single
mRNA, under control of one promoter.
• Genes in the lac operon specify proteins that
help the cell utilize lactose.
• lacZ encodes an enzyme that splits lactose into
monosaccharides (single-unit sugars) that can be
fed into glycolysis.
• Similarly, lacY encodes a membrane-embedded
transporter that helps bring lactose into the cell.

The control of the lac operon

• In addition to the three genes, the lac operon
also contains a number of regulatory DNA
sequences. These are regions of DNA to which
particular regulatory proteins can bind,
controlling transcription of the operon.

• The promoter is the binding site for RNA polymerase, the enzyme
that performs transcription.
• The operator is a negative regulatory site bound by
the lac repressor protein. The operator overlaps with the promoter,
and when the lacrepressor is bound, RNA polymerase cannot bind
to the promoter and start transcription.
• The CAP binding site is a positive regulatory site that is bound by
catabolite activator protein (CAP). When CAP is bound to this site, it
promotes transcription by helping RNA polymerase bind to the
promoter.

The lac repressor
• The lac repressor is a protein that represses (inhibits) transcription of the lac
operon.
• It does this by binding to the operator, which partially overlaps with the
promoter. When bound,the lac repressor gets in RNA polymerase's way and
keeps it from transcribing the operon.
• When lactose is not available, the lac repressor binds tightly to the operator,
preventing transcription by RNA polymerase.
• However, when lactose is present, the lac repressor loses its ability to bind
DNA. It floats off the operator, clearing the way for RNA polymerase to
transcribe the operon.
•

allolactose
• This change in the lac repressor is caused by the small
molecule allolactose, an isomer (rearranged version) of
lactose.
• When lactose is available, some molecules will be
converted to allolactose inside the cell. Allolactose binds to
the lac repressor and makes it change shape so it can no
longer bind DNA.
• Allolactose is an example of an inducer, a small molecule
that triggers expression of a gene or operon.
• The lac operon is considered an inducible operon because
it is usually turned off (repressed), but can be turned on in
the presence of the inducer allolactose.

Catabolite activator protein (CAP)
• When lactose is present, the lac repressor loses its
DNA-binding ability.
• This clears the way for RNA polymerase to bind to the
promoter and transcribe the lac operon.
• CAP isn't always active (able to bind DNA). Instead, it's
regulated by a small molecule called cyclic
AMP (cAMP). cAMP is a "hunger signal" made by E.
coli when glucose levels are low.
• cAMP binds to CAP, changing its shape and making it
able to bind DNA and promote transcription. Without
cAMP, CAP cannot bind DNA and is inactive

• CAP is only active when glucose levels are low
(cAMP levels are high). Thus, the lac operon
can only be transcribed at high levels when
glucose is absent.
• This strategy ensures that bacteria only turn
on the lac operon and start using lactose after
they have used up all of the preferred energy
source (glucose).

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.

• Glucose present, lactose absent: No
transcription of the lac operon occurs. That's
because the lac repressor remains bound to
the operator and prevents transcription by
RNA polymerase. Also, cAMP levels are low
because glucose levels are high, so CAP is
inactive and cannot bind DNA.
•

• Glucose present, lactose present: Low-level
transcription of the lac operon occurs.
The lac repressor is released from the
operator because the inducer (allolactose) is
present. cAMP levels, however, are low
because glucose is present. Thus, CAP remains
inactive and cannot bind to DNA, so
transcription only occurs at a low, leaky level.

• Glucose absent, lactose absent: No transcription
of the lac operon occurs. cAMP levels are high
because glucose levels are low, so CAP is active
and will be bound to the DNA. However,
the lac repressor will also be bound to the
operator (due to the absence of allolactose),
acting as a roadblock to RNA polymerase and
preventing transcription.
•

• Glucose absent, lactose present: Strong
transcription of the lac operon occurs.
The lac repressor is released from the operator
because the inducer (allolactose) is present.
cAMP levels are high because glucose is
absent, so CAP is active and bound to the DNA.
CAP helps RNA polymerase bind to the
promoter, permitting high levels of
transcription.

1. When lactose is absent
• A repressor protein is continuously synthesised. It sits
on a sequence of DNA just in front of the lac operon,
the Operator site
• The repressor protein blocks the Promoter site where
the RNA polymerase settles before it starts
transcribing
Regulator
gene
lac operonOperator
site
z y a
DNA
I
O
Repressor
protein
RNA
polymeraseBlocked

2. When lactose is present
• A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
• This causes the repressor protein to change its shape
(a conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site
z y a
DNA
I O

2. When lactose is present
• A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
• This causes the repressor protein to change its shape
(a conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site
Promotor site
z y a
DNA
I O

3. When both glucose and lactose are
present
• This explains how the lac operon is transcribed only
when lactose is present
• BUT….. this does not explain why the operon is not
transcribed when both glucose and lactose are
present.

• When glucose and lactose are present RNA
polymerase can sit on the promoter site but it is
unstable and it keeps falling off
Promotor site
z y a
DNA
I O
Repressor protein
removed
RNA
polymerase

4. When glucose is absent and lactose
is present
• Another protein is needed, an activator protein. This
stabilises RNA polymerase.
• The activator protein only works when glucose is
absent
• In this way E. coli only makes enzymes to metabolise
other sugars in the absence of glucose.
Promotor site
z y a
DNA
I O
Transcription
Activator
protein steadies
the RNA
polymerase

Summary
Carbohydrates Activator
protein
Repressor
protein
RNA
polymerase
lac Operon
+ GLUCOSE
+ LACTOSE
Not bound
to DNA
Lifted off
operator site
Keeps falling
off promoter
site
No
transcription
+ GLUCOSE
- LACTOSE
Not bound
to DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
- LACTOSE
Bound to
DNA
Bound to
operator site
Blocked by
the repressor
No
transcription
- GLUCOSE
+ LACTOSE
Bound to
DNA
Lifted off
operator site
Sits on the
promoter site
Transcription

Alternative RNA Structures from 5’ UTR
Termination signal due toTermination signal due to
hairpin formed by 3+4 pairinghairpin formed by 3+4 pairing
followed by string of uracilsfollowed by string of uracils
No terminationNo termination
signal formedsignal formed
Formation of termination signal depends onFormation of termination signal depends on
level of tryptophan carried by tRNA in the cell.level of tryptophan carried by tRNA in the cell.

Attenuation
Premature Termination of Transcription
Ribosome translates
trp codons, preventing 2+3 pairing
3+4 pairing forms terminator

Antitermination
Ribosome stalls at trp codons,
allowing 2+3 pairing
Transcription continues
toward trp E, D, C. B, A

Summary of Trp Operon Regulation
Level ofLevel of
TryptophanTryptophan
Trp OperonTrp Operon
LowLow
HighHigh
OnOn
Trp repressor inactiveTrp repressor inactive
Lack of attenuation leads to high rate ofLack of attenuation leads to high rate of
mRNA productionmRNA production
OffOff
Tryptophan + repressor = Active repressorTryptophan + repressor = Active repressor
Reduction of mRNA production by attenuationReduction of mRNA production by attenuation

Gene expression in eukaryotes
• In eukaryotic cells, the ability to express biologically active proteins comes
under regulation at several points:
• 1. Chromatin structure The physical structure of the DNA, as it exists
compacted into chromatin, can affect the ability of transcriptional
regulatory proteins (termed transcription factors) and RNA polymerases
to find access to specific genes and to activate transcription from them.
The presence modifications of the histones and of CpG methylation most
affect accessibility of the chromatin to RNA polymerases and transcription
factors.
• 2. Epigenetic control : Epigenesis refers to changes in the pattern of gene
expression that are not due to changes in the nucleotide composition of
the genome. Literally "epi" means "on" thus, epigenetics means "on" the
gene as opposed to "by" the gene.
Sanju kaladharan

• 3. Transcriptional initiation This is the most important mode for control of
eukaryotic gene expression (see below for more details). Specific factors
that exert control include the strength of promoter elements within the
DNA sequences of a given gene, the presence or absence of enhancer
sequences (which enhance the activity of RNA polymerase at a given
promoter by binding specific transcription factors), and the interaction
between multiple activator proteins and inhibitor proteins.
• 4. Transcript Processing and Modification: Eukaryotic mRNAs must be
capped and polyadenylated, and the introns must be accurately removed
(see RNA Synthesis Page). Several genes have been identified that
undergo tissue-specific patterns of alternative splicing, which generate
biologically different proteins from the same gene.
Sanju kaladharan

• 5. RNA Transport: A fully processed mRNA must leave the nucleus in
order to be translated into protein.
• 6. Transcript Stability: Unlike prokaryotic mRNAs, whose half-lives
are all in the range of 1 to 5 minutes, eukaryotic mRNAs can vary
greatly in their stability. Certain unstable transcripts have sequences
(predominately, but not exclusively, in the 3'-non-translated regions)
that are signals for rapid degradation.
• 7.Transalational initiation Since many mRNAs have multiple
methionine codons, the ability of ribosomes to recognize and initiate
synthesis from the correct AUG codon can affect the expression of a
gene product. Several examples have emerged demonstrating that
some eukaryotic proteins initiate at non-AUG codons. This
phenomenon has been known to occur in E. coli for quite some time,
but only recently has it been observed in eukaryotic mRNAs.
Sanju kaladharan

• 8. Small Rna mediated Within the past several years a new model of
gene regulation has emerged that involves control exerted by small
non-coding RNAs. This small RNA-mediated control can be exerted
either at the level of the translatability of the mRNA, the stability of
the mRNA or via changes in chromatin structure.
• 9. Post transalational activation Common modifications include
glycosylation, acetylation, fatty acylation, disulfide bond formations,
etc.
• 10. Protein Transport: In order for proteins to be biologically active
following translation and processing, they must be transported to
their site of action.
• 11. Control of Protein Stability: Many proteins are rapidly degraded,
whereas others are highly stable. Specific amino acid sequences in
some proteins have been shown to bring about rapid degradation.
Sanju kaladharan

HDACS
• Histone Deacetylases (HDACs)
• HDACs are responsible for removing the acetyl groups put on histones
(and other proteins) by the histone acetyltransferases (HATs). This process
is a vital aspect of epigenetic regulation of gene expression and more
generally for the control of cellular stability.
/
Sanju kaladharan

DNA BINDING PROTEINS
• DNA-binding proteins are proteins composed
of DNA-binding domains and thus have a
specific or general affinity for either single or
double stranded DNA
Sanju kaladharan

GENE REGULATION IN PROKARYOTES AND EUKARYOTES

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