Regulation of Gene Expression
in Bacteria
MB 206 : MODULE 1 - B
Angelia Teo Jan 09
Regulation of Gene Expression
 A cell contains the entire genome of an organism–
ALL the DNA.
 Gene expression = transcribing and translating the
gene
 Regulation allows an organism to selectively
transcribe (and then translate) only the genes it
needs to.
 Genes expressed depend on
 the type of cell
 the particular needs of the cell at that time.
Principles of Gene Regulation
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How Are Genes Regulated?
 Genes located in coherent packages called
operons
 operons has 4 parts
 regulatory gene - controls timing or rate of
transcription
 promoter - starting point
 operator - controls access to the promoter by RNA
polymerase
 structural genes
 NOTE = operons regulated as units
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Gene Regulation in Prokaryotes
 Prokaryotes organize their genome into operons
 Operon = a group of related genes
 One promoter sequence at the very beginning
 All of the genes will be transcribed together (in one long
strand of RNA.
Principle of Gene Regulation
 RNA polymerase binds to DNA at promoters.
 Transcription initiation is regulated by proteins that bind to or near
promoters.
 Repression of a repressible gene: (i.e., negative regulation)
repressors (vs activitors) bind to operators of DNA.
 Repressor is regulated by an effector, usually a small molecules or a
protein, that binds and causes a conformational change.
 Activitor binds to DNA sites called
enhancer to enhance the RNA
polymerase activity.
(i.e., [positive regulation)
 Induction of an inducible gene, e.g., heat-shock genes.
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General organization of an inducible gene
Regulation of Genes
Transcription Factor
(Protein)
RNA polymerase
DNA
Regulatory Element
Gene
Regulation of Genes
New protein
RNA
polymerase
Transcription Factor
DNA
Regulatory Element
Gene
Gene Expression
How much protein is in a cell (and active)??
Some gene products are
necessary only some of
the time
Response to
environmental
changes (eg.
Temperature)
Response to life
events (eg.
Reproduction)
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Most genes are not expressed at
a particular time
 Not all of the genes in a bacteria will be
expressed at the same time.
 Even in some of the smallest bacteria, about 500
different genes exists
 Of the 4279 genes in E. coli , only about 2600 (~60%)
are expressed in standard laboratory conditions.
 Only about 350 genes are expressed at more than 100
copies (i.e. molecules!) per cell, making up 90% of the
total protein.
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Possible target in control of gene expression
Topics:
 Lac Operon (Negative control & Catabolic repression)
 Tryptophan Operon (Positive control)
 Histidine Operon (Attenuator)
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Comparison of genomes of various organism
ORGANISM
%coding
Size of genome
(bp)
number of
genes
Escherichia coli
90%
4,639,221 bp
4288
Mycoplasma genitalium
88%
580,073 bp
468
Haemophilus influenzae
86%
2,087,778 bp
1,662
Methanococcus jannaschii
85%
1,660,000 bp
1,997
Synechocystis sp.
(PCC 6803)
80%
3,570,000 bp
3,168
Saccharomyces cerevisiae
~75%
13,000,000 bp
6,275
Humans
~2%
3,000,000,000 bp
70,000 (?)
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Diploid numbers of some commonly studied organisms
(as well as a few extreme examples)
Homo sapiens (human)
46
Mus musculus (house mouse)
40
Drosophila melanogaster (fruit fly)
8
Caenorhabditis elegans (microscopic roundworm)
12
Saccharomyces cerevisiae (budding yeast)
32
Arabidopsis thaliana (plant in the mustard family)
10
Xenopus laevis (South African clawed frog)
36
Canis familiaris (domestic dog)
78
Gallus gallus (chicken)
78
Zea mays (corn or maize)
20
Muntiacus reevesi (the Chinese muntjac, a deer)
23
Muntiacus muntjac (its Indian cousin)
6
Myrmecia pilosula (an ant)
2
Parascaris equorum var. univalens (parasitic
roundworm)
2
Cambarus clarkii (a crayfish)
20
0
Equisetum arvense (field horsetail, a plant)
21
6
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Genes in E.coli
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E. coli genes expressed
 A total of 4288 genes in Escherichia coli
- 2600 genes found under standard laboratory growth
conditions
- 2100 protein spots detected under 2-D protein gels
- 350 proteins in high amount, the rest are very low
amounts
 Majority of the genes are likely to be expressed transiently,
in small amounts during DNA replication, and then remain
silent (unexpressed) until the next round of DNA synthesis
Why is there a need to control gene expression?
1) Prevent energy wastage
2) Ensure only necessary proteins are made
according to the requirement for cells growth.
• Small portion of DNA in cell used for genetic
message (mRNA), the rest for regulatory purposes.
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Gene Regulation in bacteria
 How do single-celled prokaryotes like E. coli know how
to respond to their environments?
Each environmental cue generates a specific
response, with specific proteins and reactions.
eg.
A bacterium can use different sources of Nitrogen
- incorporate N2 gas from the air
- incorporate ammonia from their surroundings or
- from amine group of an amino acid like glutamine
(easier and less energy)
These processes involve very different enzymes. Presence of glutamine, the
cell will turn off synthesis of enzymes for fixing N2
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How can the cell "turn off" the synthesis of
proteins from its DNA?
Expression of genes in microbes is often regulated by
intracellular or environmental conditions.
Many different types of regulation
Most genes are regulated at the point of transcription
initiation – early intervention control
Regulation at different stage of gene expression:
• - transcription initiation or termination – no production of mRNA
• - mRNA stability or translation into protein.
• - protein modification and stability - prevent wasteful reactions.
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Gene regulation can occur at any place along the flow of information
from DNA to RNA to protein:
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Different forms of gene regulation
a. Regulation by DNA Replication (default)
b. Transcriptional Regulation by different s-factors.
c. Negative Regulation of Gene Expression
d. Positive Control of Gene Regulation
e. Alternative splicing of RNA (almost exclusively for eukaryotes)
f. Post-transcriptional regulation
- termination of transcription
- translation control
- message stability
- protein stability
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E.coli RNA Polymerase subunits
Gene
Mass
KDa
Use
-35 Sequence
separation
-10 Sequence
rpoA
40
a subunit
-
-
-
rpoB
155
b subnit
-
-
-
rpoC
160
b' subunit
-
-
-
rpoD
70
s70
General
TTGACA
16-18 bp
TATAAT
rpoN
54
s54
Nitrogen
CTGGNA
6 bp
TTGCA
rpoS
38
s38
Stationary
not known
not known
not known
rpoH
32
s32
Heat shock
CCCTTGAA
13-15 bp
CCCGATNT
fliA
28
s28
Flagellar
CTAAA
15 bp
GCCGATAA
24
s24
High temp.
heat shock
not known
not known
not known
rpoE
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Transcription regulation by s-factors
 s70 - RpoD
s54 - RpoN
s38 - RpoS
s32 - RpoH
s28 - FliA
s24 - RpoE
“normal” s-factor
Nitrogen response
Stationary phase
Heat shock response
Flagellar genes regulation
Heat shock high temp.
 Approx: 1500 - 2000 copies of RNAP holoenzyme/ cell
 For bacteria growing in "log phase":
 ~600 copies of RpoD (s70)
 ~200 copies of RpoS (s38)
 [RpoS] increases to ~600 copies per cell in stationary phase or
osmotic shock.
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Operon and Regulon
 An operon
- consists of a set of genes expressed coordinately & transcribed
as a single unit
- Specific regulation (positive / negative) can induce or repress a
particular gene or operon
- contains both a regulatory & a message region.
- Regulatory / control region at the 5’ side of the gene & codes for a
protein (message region).
 Regulon
- comprise of global regulation affecting a set of operons.
- All operons in the regulon are coordinately controlled by the
same regulatory mechanism.
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 Regulated genes can be switched on and
off depending on the cell’s metabolic
needs
 Operon : a regulated cluster of adjacent
structural genes, operator site, promotor
site, and regulatory gene(s)
Repressible vs. Inducible Operons
two types of negative gene regulation
Repressible Operons
 Genes are initially ON
 Anabolic pathways
 End product switches
off its own production
Inducible Operons
 Genes are initially OFF
 Catabolic pathways
 Switched on by
nutrient that the
pathway uses
The lac Operon of E. coli
1. Growth and division genes of bacteria are regulated genes. Their expression is
controlled by the needs of the cell as it responds to its environment with the
goal of increasing in mass and dividing.
2. Genes that generally are continuously expressed are constitutive genes
(housekeeping genes). Examples include protein synthesis and glucose
metabolism.
3. All genes are regulated at some level, so that as resources dwindle the cell can
respond with a different molecular strategy.
4. Prokaryotic genes are often organized into operons that are cotranscribed. A
regulatory protein binds an operator sequence in the DNA adjacent to the gene
array, and controls production of the polycis-tronic (polygenic) mRNA.
5. Gene regulation in bacteria and phage is similar in many ways to the emerging
information about gene regulation in eukaryotes, including humans. Much
remains to be discovered; even in E. coli, one of the most closely studied
organisms on earth, 35% of the genomic ORFs have no attributed function.
Chapter 16 slide 29
台大農藝系 遺傳學 601 20000
The lac Operon of E. coli
Animation: Regulation of Expression of the lac Operon
Genes
1. An inducible operon responds to an inducer substance
(e.g., lactose). An inducer is a small molecule that joins
with a regulatory protein to control transcription of the
operon.
2. The regulatory event typically occurs at a specific DNA
sequence (controlling site) near the protein-coding
sequence (Figure 16.1).
3. Control of lactose metabolism in E. coli is an example of
an inducible operon.
Chapter 16 slide 30
台大農藝系 遺傳學 601 20000
Lac Operon
 Transcription is “OFF”
 When there is no lactose that needs to be
digested
 lacI repressor is in active form  binds to
operator  blocks RNA Polymerase  no
transcription
Lac Operon
 Transcription is “ON”
 When there is lactose that needs to be digested
 Lactose binds to lacI repressor  inactivates it
 RNA Polymerase is able to bind to promoter 
transcribe genes
Negative Regulation of Gene Expression
 By default, the gene is usually
switched ON.
 Binding of a REPRESSOR will switch
the gene OFF.
 Most common regulation in BACTERIA
 Often this is found as AUTOREGULATION - where too much
of the gene product inhibits further transcription - usually
this is through binding to the upstream promoter control
region.
 A good "classic" example is the E.coli lac operon.
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Positive control of Regulation
 By default, the gene is usually
switched OFF.
 Binding of a ACTIVATOR will switch
the gene ON.
(often transcriptional activators / factors
bind and bend DNA upstream of the
promoter.)
 Most common in EUKARYOTES
 Some promoters are not very functional in the absence of a
transcriptional activator protein(s).
Angelia Teo Jan 09
Lac Operon
 Lactose metabolism occurs when the environment contains lactose.
 Enzymes required for lactose degradation are TURNED ON.
beta-galactosidase (lac Z)
- enzyme hydrolyzes the bond between glucose & galactose.
Lactose Permease (LacY)
- enzyme spans the cell membrane
- transports lactose into the cell from the outside environment.
- Membrane is otherwise essentially impermeable to lactose.
Thiogalactoside transacetylase (LacA)
- The function of this enzyme is not known.
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Lactose metabolism
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Regulatory elements in the Lac Operon
Element
Function
Operator (LacO)
binding site for repressor
Promoter (LacP)
binding site for RNA polymerase
Repressor (LacI)
Pi
CAP
codes for lac repressor protein
Binds to DNA at operator and blocks
binding of RNA polymerase at promoter
promoter for Lac I
binding site for cAMP/CAP complex
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Glucose or Lactose?
 A bacterium's prime source of food is glucose, since it does not
have to be modified to enter the respiratory pathway.
 So if both glucose & lactose are around, the bacterium will
to turn off lactose metabolism in favor of glucose metabolism.
 There are sites upstream of the Lac genes that respond
to different glucose concentrations.
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Presence of inducer - lactose
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Regulation of Lac operon depending on availability of lactose or glucose
Absence of lactose
Low levels of Glucose /
Catabolite repression
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Regulation of Lac Operon
 When lactose is present, it acts as an inducer of the operon. Lactose enters
the cell and binds to the Lac repressor, inducing a conformational change
preventing the repressor from binding to the operator. This allows the RNA
polymerase binding at the promoter to proceed with transcription of mRNA
(LacZ, LacY & LacA) and production of enzymes for the metabolism of
lactose.
 When the inducer (lactose) is removed, the repressor returns to its original
conformation and binds to operator, blocking the RNA polymerase from
proceeding with transcription of mRNA, thus no protein is made.
 The lac operon is always primed for transcription upon the addition of
lactose.
 When levels of glucose (a catabolite) in the cell are high, formation of cyclic
AMP is inhibited. But when glucose levels drop, more cAMP forms. cAMP
binds to a protein called CAP (catabolite activator protein), which is then
activated to bind to the CAP binding site. This activates transcription, by
increasing the binding affinity of RNA polymerase to promoter. This is called
catabolite repression, a misnomer since it involves activation, but
understandable since it seemed that the presence of glucose repressed all
the other sugar metabolism operons.
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The Tryptophan Operon (Positive regulation)
 Trp operon contains the Tryptophan biosynthetic genes.
 Trp repressor protein can bind to the operator of Trp operon
 When tryptophan is high, it binds to the repressor and induces a
change so that the repressor can now bind to DNA.
 When tryptophan are low in the cell, tryptophan falls off the
repressor, and the repressor goes back to its original
conformation, losing its ability to bind to the DNA. RNA
polymerase binds to the promoter and transcription proceeds,
making tryptophan biosynthetic genes and replenishing the
cell's supply of tryptophan.
 This type of feedback inhibition of transcription is very common.
ribosomal RNA can also act to repress their own synthesis.
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Repressible Operon: Trp Operon
 Repressible Operon = Operon that is usually “ON”
but can be inhibited
 The Trp Operon
 example of a repressible operon
 Genes that code for enzymes needed to make the amino
acid tryptophan
TrpR Gene
 TrpR gene is the regulatory gene
for the Trp operon
 Found somewhere else on the
genome
 NOT part of the Trp operon
 TrpR gene codes for a protein = TrpR
repressor
 TrpR gene is transcribed and
translated separately from the Trp
operon genes.
TrpR Repressor
 Repressor protein is translated in an inactive
form
 Tryptophan is called a corepressor
 When tryptophan binds to the TrpR repressor, it
changes it into the active form
Operator Region
 There is also an operator region of DNA in the Trp
Operon
 Just after the promoter region
 The TrpR Repressor can bind to the operator if it’s in the
active form
Trp Operon
 Transcription is “ON”
 Occurs when there is no tryptophan available to
the cell.
 Repressor is in inactive form (due to the absence
of tryptophan)
 RNA Polymerase is able to bind to promoter and
transcribe the genes.
Trp Operon
 Transcription is “OFF”
 Occurs when tryptophan
is available
 Tryptophan binds to the
TrpR repressor 
converts it to active form
 TrpR protein binds to
operator  blocks RNA
Polymerase  no
transcription
The Histidine Operon (An Attenuator)
 The histidine operon functions in a slightly different way.
 At the beginning of the operon there is a leader coding region
AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCUGAC
Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-ProAsp
When transcription begins, the RNA comes of the DNA and ribosomes
hop onto it to start translation.
 Low amount of histidine in the cell:
- the ribosome stalls because no aminoacyl tRNA's charged with histidine.
- this leaves a long stretch of RNA (for RNA ploymerase is still transcribing
it) with no ribosomes bound to it.
Angelia Teo Jan 09
The Histidine Operon (An Attenuator)
 High amount of histidine in the cell:
- the ribosome is not stalled
- the leader sequence in RNA allows it to form a terminator loop
(attenuation site), at which point the RNA is cleaved
- RNA polymerase stops transcribing the genes
- the terminator only functions when the ribosome is not stalled.
 Many amino acid synthetic operons are also controlled by some form of
attenuation. The tryptophan operon has both attenuation control and
repressor control.
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Methods for Studying Regulation


Possible mutations in various elements of the Lac operon, give rise to
mutants
How to study the lac operon? Tools used:
 IPTG (isopropyl-beta-D-thiogalactoside)
- a molecule analogue to lactose, binds to the Lac repressor (Lac I).
- used as a gratuitous inducer to induce Lac operon
- but not a substrate for the lactose metabolism genes.
 Spectrophotometer quantification of B-galactosidase activity.
- Quantify amount of mRNA made (coding lacZ, lacY, and lacA)
- B-galactosidase can cleave a colourless substrate called ONPG into a
yellow product , ONP - quantitated by spectrophotometer.
- The degree of yellowness - indicates enzyme activity or amount of
transcription of mRNA or the activity of lac operon.
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Methods for Studying Regulation
 Different types of gene expression:
- Constitutively ( c ) expressed gene is never turned off, it is
making mRNA and protein all the time.
- Inducible gene can be turned on by an inducer.
- Uninducible gene is never turned on. DNA binding site is
mutated preventing binding by an inducer.
- Super-repressor ( s ) always represses, regardless
of its regulation. eg. a Lac I (s) mutant always represses the
promoter whether or not lactose is present.
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Cis or Trans-regulation
 DNA element 1 and DNA element 2
How to determine whether DNA element 1 is acting in cis or in
trans to one another ?
Test: insert a piece of DNA carrying DNA element 1 into a cell that already has a copy
of mutated DNA element 1 adjacent to DNA element 2.
A) Observation: the cell recovers it’s function.
Conclusion: The inserted element can complement or replace the function of the
mutated element 1, it can be said to be trans acting, since it must diffuse off a
plasmid or from another site in the DNA in order to be functional. This, therefore
involves a diffusible protein.
B) Observation: the cell does not regains it’s function
Conclusion: the two functional pieces of DNA must be adjacent to each other to be
functional (cis acting),
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BLA OPERON
- Control of gene expressing resistance to Penicillin
 Usually present on a plasmid in S aureus;
 its function is B-lactam-induced production of penicillinase.
 Bla operon composed of 3 genes:
blaZ = codes for a penicillin-hydrolysing enzyme (penicillinase)
blaR1 & bla l = transcription regulator genes
 When penicillin is in the environment, membrane-bound signal
transducer protein BlaR1 recognises it and transmits the signal to
the cytoplasm.
 Repressor protein Bla I, which binds near to the promoter of blaZ
preventing its transcription, is cleaved off.
 blaZ is transcribed efficiently to produce penicillinase.
Angelia Teo Jan 09
Negative regulation:
Substrate induction
Positive
regulation of
the lac
operon
Positive Gene Regulation
 In the lac operon there are other molecules to
further stimulate transcription.
 Lactose will only be digested for energy when
there isn’t much glucose around
 When glucose levels are low, level of cAMP
molecule builds up
cAMP and CAP
 CAP = regulatory protein
that binds to cAMP
 CAP is inactive unless cAMP
binds to it
Positive gene regulation
 If there isn’t much
glucose high levels
of cAMP
 CAP and cAMP bind 
CAP can bind to the
promoter  stimulates
RNA Polymerase to
bind
Positive gene regulation
 When glucose levels rise again, cAMP levels
will drop  no longer bound to CAP
 CAP can’t bind to promoter  transcription
slows down
Positive gene regulation
 The lac operon is controlled on 2 levels:
 Presence of lactose determines if transcription can
occur
 CAP in the active form determines how fast
transcription occurs
Lac Operon
No Food
Milk
Glucose?
Low: cAMP
high, CRP
attaches
Lactose?
Low:
Repressor
attached
Glucose?
Low: cAMP
high, CRP
attaches
Lactose?
Low:
Repressor
removed
Lac Operon
Milkshake
Powerade
Glucose?
High: cAMP
low, CRP off
Lactose?
Low:
Repressor
removed
Glucose?
High: cAMP
low, CRP off
Lactose?
Low:
Repressor
attached
POWERade is a drink manufactured by
The Coca-Cola Company.
Lac Operon
No food
Milk
• ready to be enhanced but off
• enhanced transcription
Milkshake
• un-enhanced transcription
Powerade
• off and un-enhanced
Induction by negative or positive control
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Negative regulation: end-product repression