Regulation of gene

expression in
Prokaryotic cells

Ratchada Cressey, Ph.D

Assistant Professor
Clinical Chemistry
Associated Medical Science
Chiang Mai University
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Comparison of Prokaryotic and
Eukaryotic cell structure

Prokaryotic Cell (Bacillus megaterium)

Eukaryotic Cell (L-Cell)

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Comparison of Prokaryotic and
Eukaryotic gene structure

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 Logic of gene expression
Eukaryotes
• Eukaryotes are (mostly) metazoan
– Colonies of specialized cells
– Almost all cells die at the end of generation
– Only gamates survive- they do not respond
to environmental stimuli
• Most cells provide a specialized function
• Few cells are involved in responses to
environmental stimuli

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 Logic of gene expression
Prokaryotes
• Cells respond to fast environmental
changes
• Must compete for carbon sources
• Changes in gene may ‘persist’ for
several generations
• Gene expression is capable of
responding to signals not seen in many
generations

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Gene Regulation in Bacteria
• Bacteria adapt to changes in their surroundings by
using regulatory proteins to turn groups of genes on a
nd off in response to various environmental signals.

• The DNA of Escherichia coli is sufficient to encode about

4000 proteins, but only a fraction of these are made at a
ny one time. E. coli regulates the expression of many of its g
enes according to the food sources that are available to it.

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Gene Structure of bacteria

Transcription
Transcription start site

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 E. coli Promoters

consensus

TATA (Pribnow) box

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Gene expression is regulated in many
different ways in prokaryotes

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Method for studying of DNA-
protein interaction
• EMSA (Electrophoresis Mobility
Shift Assay) or gel shift assay
• DNA footprinting
– DNAse I footprinting

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Gel shift assay

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2. DNAse I Footprinting

1. Prepare end-labeled
DNA.
2. Bind protein.
3. Do a mild digestion with
DNAse I (Dnase I
randomly cleaves DS
DNA on each strand)
4. Separate DNA
fragments on
denaturing acrylamide
gels (sequencing gels)
5. Expose gel to X-Ray
film.
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Fig. 5.37a
Sample of a DNase I Fig. 5.37b
footprinting gel (for a
DNA-binding protein).

Footprint

Samples in lanes 2-4

had increasing
amounts of the DNA-
binding protein
(lambda protein cII);
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lane 1 had none.
Differences between prokaryotes
and eukaryotes:

• Prokaryote gene expression typically is regulated

by an operon, the collection of controlling sites
adjacent to protein-coding sequence.

• Eukaryotic genes also are regulated in units of

protein-coding sequences and adjacent
controlling sites, but operons are not known to
occur.

• Eukaryotic gene regulation also is more complex

because eukaryotes possess a nucleus.
(transcription and translation are not coupled).
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 What is An Operon?
• As we have learned, Operons aid or
repress the transcription proteins.
• Operons are made up of the Operator, and
the genes it controls.
• Operons are synthesized into a single
molecule of mRNA that holds the
information for the Prokaryote to
transcribe.

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How do Operons Regulate?
• Positive • Negative
Control – Control – mRNA
Operons only synthesis proceeds
function in the more rapidly in the
presence of a absence of the
controlling factor. active controlling
factor.

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Induction and Repression of
bacterial enzyme

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 A Little History First….
• In 1961, Jacob and Monod found a
protein, a repressor, that could control the
production of β -galactosidase. They
believed that this protein worked when
bonded to an operator. They named this
complex the Lac Operon, and won the
Nobel Prize in 1964.

François Jacob Jacques Monod

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 1. Inducible System
• Inducers – raise the levels of inducible
enzymes.
• Repressor Proteins – repress mRNA
synthesis, this is the active control factor.
• Inducers – Bind with the repressor,
making it inactive and allowing
transcription to take place, to create, the
inducible enzyme.
No Inducers = No Enzymes = No
Metabolism
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 The Lactose Operon
• Negative Control System in E. Coli
• Gives a good example of a Inducible
System.

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Regulation of the Lac operon

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The Lac Inducible System
Negative control

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 How does this inhibit
transcription?
• The Promoter, where the RNA polymerase
binds is located next to the Operator,
when a repressor binds to it, it bends the
DNA so the RNA polymerase will not bind
or can not begin to transcribe.

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The binding of repressor to the
Lac operon

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The Lac Inducible System
Repressor is Turned Off

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•Lactose itself was not the inducer but several galactosides which are
not metabolized would work.
•allolactose was found to be the physiological inducer. It is a secondary
metabolite of lactose as a byproduct of basal ß-galactosidase activity 28
IPTG (isopropylthiogalactoside) is a good ,
non-metabolized inducer. 29
 What about Positive
Control?
• Operons function when the
controlling factor is present.
• The Lac Operon is also good example
of Positive control.

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 Adenylate cyclase and CAP mediate glucose repression of Lac
Adenylate cyclase (AC) is an enzyme that synthesizes cyclic AMP (cAMP) from ATP

AC

AMP glucose cAMP

High glucose ⇒ adenylate cyclase is inhibited (indirectly, via a catabolic

product) Therefore cAMP levels are LOW

Absence of glucose ⇒ adenylate cyclase is NOT

repressed. Therefore cAMP levels are HIGH

cAMP forms a complex with the CAP protein, which allows it to then bind to the CAP site
upstream of the Lac operon. Binding of the CAP protein is required to allow RNA
polymerase to bind to the lac promoter and turn on transcription. In the absence of CAP
binding, there is no (or very little) transcription of the lactose operon, even in the presence
of lactose. 32
cAMP Regulartory Protein (CRP)

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cAMP Regulartory Protein (CRP)

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Interaction of cAMP, CAP, and the Lac Repressor

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 cAMP binding causes
conformational change of CRP

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CAP mediates glucose repression of Lac

Promotes transcription

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 Four States of the Lac Operon
LacI
Lactose Glucose
- +

- -
CAP-cAMP

+ -

+ +
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 2. Repressible System
• Aporepressor – the inactive form of a
repressor.
• Corepressors – bind to the aporepressor,
and make it an active repressor.
• Repressible Systems – enzymes are
reduced by the presence of the end
product.
• Good example is the Tryptophan operon.

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 Tryptophan operon:
Regulation by repression and attenuation
• Genes for tryptophan synthesis
• Repressed by end-product of pathway,
Tryptophan.
• Repression requires Operator sequence,
Aporepressor (trpR gene product) & Co-
repressor (Tryptophan).

• Also controlled by attenuation in the “Leader”

peptide region of the transcript.
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Tryptophan operon

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Tryptophan Operon

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Attenuation provides secondary control
mechanism in the Trp operon

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Attenuation provides secondary control
mechanism in the Trp operon
• Attenuation – the premature termination of
transcription.
• Leader Region – lies between the Operator and the
1st structural gene. It contains four segments we
will call 1, 2, 3, 4.

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 Tryp operon: Repressible control
• Segment 1 contains 2 trp codons.
• If tryptophan levels are low, translation in
segment 1 are slow therefore segment 2 is not
bound by ribosomes, and is free to hairpin with
segment 3, and transcription occurs.
• If trytophan levels are high, the tryptophanyl-
tRNA is available for proteins synthesis,
ribosomes will to bind with segment 2, therefore
3 and 4 hairpin to create a termination site.

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 The trp Leader peptide has two key tryptophan codons.

The ribosome stalls at the trp codons when [Tryptophan]

is too low.
The stalled ribosome prevents a downstream
transcription terminator (IR + U-rich sequence) from
forming.
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Fig. 7.35
Fig. 7.36 49
Mechanism of Attenuation

• rU-dA base-pairs
are exceptionally
weak, they have
melting temperature
20C lower than rU-
rA or dT-rA

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Attenuation vs. No Attenuation

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 The Arabinose Operon
• Ara operon: 3 structural genes required to utilize the
sugar arabinose (araB, araA and araD).

• Regulator protein ⇒ araC

• AraC turns on transcription of the ara operon by binding

to the araI initiatior site only when it is bound to arabinose

• In the absence of arabinose, araC protein undegoes a

conformation change ⇒ Now binds to BOTH araI and araO,
which forms a loop that inhibits transcription.

• The Ara operon is also subject to catabolite repression.

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Arabinose Operon

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 The Arabinose Operon
Arabinose present, Glucose absent, operon ON

No Arabinose present, operon OFF

This loop prevents RNA transcription (NOT true for all loops) 55
The Arabinose Operon

RNA polymerase

AraC

CAP-cAMP Arabinose

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 Autoregulation of araC

• As the level of AraC (green) rises, it binds to araO1 and prevent

transcription leftward from Pc through the araC gene, thus preventing an
accumulation of too much repressor

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 Promoters and Sigma Factors

• Part of the RNA polymerase enzyme that

recognizes the promoter is called the sigma factor
. After transcription begins, this unit dissociates fr
om the enzyme
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 Different sigma factors recognize
different promoters

• Different sigma factors recognize different promoters and

thus, the availability of sigma factors can regulate the
transcription of genes associated with these promoters.

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Example of Translational Control in
Prokaryotes: Antisense RNA

• Normally, mRNA is synthesized off of the template

(antisense) strand of DNA. Antisense RNA is synthesized fro
m the noncoding (sense) strand of DNA. The two mRNA mol
ecules bond together, inactivating the mRNA
• This mechanism appears to be universal among bacteria. It
has not been shown to be a normal means in eukaryotes
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 Suggested reading:

• Robert F. Weaver, Molecular biology,

second edition, 2002

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