CM145/CM245

Molecular Biotechnology for Engineers

Prokaryotic Gene Expression Regulation

http://etc.ch/qBgb
http://etc.ch/qBgb
 Hemicellulose is an abundant feedstock

Aristilde et al. Applied and Environmental Microbiology. 81:1452-62 (2015).

How can you tell if cells can consume
multiple nutrients?

Aristilde et al. Applied and Environmental Microbiology. 81:1452-62 (2015).

When E. coli is given glucose and maltotriose,
Culture density

Hours
Bren et al. Scientific Reports. 6:24834 (2016).
 Overview of Transcriptional Regulation

Activator
Binding Site

Activator
Binding Site

(weak promoter)

Activator recruits RNA polymerase Repressor blocks RNA polymerase

 Why Regulate Gene Expression?
• Energy conservation
• Fine-tuning of cellular functions
• Control over time-dependent activities (e.g., cell
division)
• In multicellular organisms, control over tissue/cell-
type–specific activities
• Environmental adaptation (sense and respond)
 Terminology
• Activator: a DNA-binding protein that increases transcription
by recruiting RNA polymerase
• Repressor: a DNA-binding protein that decreases transcription
by blocking RNA polymerase
• Inducer site or activation site: DNA sequence to which
activator protein binds
• Operator: DNA sequence to which repressor protein binds
• Inhibitor: a molecule that “deactivates” activator proteins
(note the word “inhibitor” can mean other things in other contexts)

• Inducer: a molecule that either

o binds to and “activates” activator proteins, or
o binds to and “deactivates” repressor proteins

• Co-repressor: a molecule that binds to and “activates”

repressor proteins
 Activation and Deactivation

Here, Act is an activator

and E is an inhibitor.

• The inhibitor, inducer, or co-repressor is usually a small molecule, but it

can also be a macromolecule (but generally not a protein).

• Allosteric change: change in the properties (usually including shape) of

a protein following the binding of another molecule to the protein
Repression, Derepression, and Corepression

Here, R is a repressor
and E is an inducer.

Here, IR is an inactive
repressor and C is a
co-repressor.
 Transcriptional Regulation

A B C D E F G

Where is the…
1. Promoter
2. RBS
3. ORF
4. Start codon
5. Stop codon
6. Terminator
7. Operator site
8. Inducer site
 Transcriptional Regulation

A B C D E F G

7/8 1 7/8 2 3 2 3 6
4 5 4 5
Where is the…
1. Promoter
2. RBS
3. ORF
4. Start codon
5. Stop codon
6. Terminator
7. Operator site
8. Inducer site
 The Promoter Concept

• Housekeeping genes: Genes for products that are required at all times
• Constitutive expression: Unvarying gene expression (i.e., always ON)
• Regulated gene expression: Expression levels rise and fall in response to
molecular signals
o Inducible expression: Gene expression that would increase under particular
molecular circumstances (i.e., can be turned ON)
o Induction: The process of increasing the expression of inducible genes
o Repressible expression: Gene expression that would decrease under
particular molecular circumstances (i.e., can be turned OFF)
o Repression: The process of decreasing the expression of inducible genes
 Example of Housekeeping Gene: RuBisCO
• Ribulose-1,5-bisphosphate
carboxylase oxygenase
• Involved in the first major
step of carbon fixation in
plants
• Responsible for
transformation from the
“lifeless” to the “living”
• The most abundant protein
on earth
• For obvious reasons, a
housekeeping gene
 Example of Regulated Gene: Lac Operon
• LacZ, LacY, and LacA are all proteins involved in
lactose catabolism
• Glucose is the preferred food source
• Lactose is only catabolized when glucose is
unavailable
• How would you regulate the expression of LacZ,
LacY, and LacA?
 The Players
Pol-σ70

I O lacZ lacY lacA Terminator

+ cAMP
• cAMP production  when glucose
CAP activator level 
• Activator binding to inducer site (I)
induces expression
+ lactose
• Repressor binding to operator (O)
Lac I inactive represses expression
(repressor) repressor
 The Scenes
– lactose, + glucose (low cAMP)

Pol-σ70

No transcription

I O lacZ lacY lacA Terminator

+ lactose, + glucose (low cAMP)

Pol-σ70 Low transcription

I O lacZ lacY lacA Terminator

+ lactose, – glucose (high cAMP)

Pol-σ70 High transcription

I O lacZ lacY lacA Terminator

The Outcome: Diauxic Growth

http://cronodon.com/BioTech/Bacteria_Growth.html
 Can we make cells use mixed substrates?

Park et al. Nature Metabolism. 1:632-651 (2019).

 Can we make cells use mixed substrates?

Fed-batch bioreactor setup:

Park et al. Nature Metabolism. 1:632-651 (2019).

 Can we make cells use mixed substrates?

Park et al. Nature Metabolism. 1:632-651 (2019).

 Converting CO2 to Fuel
CO2 Acetate Lipids

H2/CO2
mix cell air cell
growth recycle recycle
medium acetate spent
medium

lipid
accumulation

Gas-to-acetate Acetate-to-lipid
conversion by conversion by
Moorella thermoacetica Yarrowia lipolytica
Park et al. Nature Metabolism. 1:632-651 (2019).
 Example #2: Arabinose Operon
• Arabinose is an alternative energy source for E. coli when
glucose is unavailable
• The ara operon encodes 3 enzymes required for arabinose
catabolism (AraB, AraA, AraD)
• Want to transcribe araBAD only when glucose is low and
arabinose is present
Activator
From Voet and Voet
Figure 29-27

L-arabinose
Repressor

araC mRNA
Control
sites
Structural genes

Regulatory gene araBAD mRNA

 Negative Regulation
• AraC Regulation
o When AraC is present, it can bind the araO1 site and block further araC
transcription
o When AraC protein is absent, araC gene is transcribed and araBAD is
also transcribed at low, basal levels
• When arabinose is absent and/or glucose is high
o Nothing binds to CAP-binding site → lack of induction
o AraC by itself binds to araI and araO2 → loops the DNA → blocks
araBAD transcription

RNA polymerase From Voet and Voet

Figure 29-28
araO2 araC CAP araI1 araI2
araBAD
araO1

araC mRNA araBAD mRNA (basal level)

araC
araO2

araO1 CAP araI1 araI2

araBAD
 Positive Regulation
• When arabinose is present AND glucose is low
o AraC binds arabinose, then the complex binds to araI and araO1
o Because araO1 is bound, araC transcription is blocked
o Arabinose-bound AraC has no affinity for araO2 → no looping
o cAMP is produced, binds to CAP, then the complex binds the CAP-
binding site
o araBAD transcribed

CAP-cAMP

AraC-arabinose AraC-arabinose
RNA polymerase
araO2 araC araO1 CAP araI1 araI2
araBAD

araBAD mRNA

• Video: http://www.youtube.com/watch?v=EVmnIDc0K7o&noredirect=1
 PBAD

araC araO2 araO1 CAP araI

PC

• araO1 is an operator site that regulates araC transcription. AraC (with or without
arabinose) binds to this site and represses its own transcription from the PC
promoter.

• araO2 is also an operator site. AraC (no arabinose) can simultaneously bind to
araO2 and the araI site to repress transcription from the PBAD promoter.

• araI is a mixed-function site. AraC (no arabinose) can simultaneously bind to

araO2 and the araI site to repress transcription from the PBAD promoter.
However, [AraC + arabinose] bound at this site helps to activate expression of
the PBAD promoter.

• CAP-cAMP binds to the CAP binding site. In conjunction with [AraC +

arabinose], it assists RNA polymerase in binding to the PBAD promoter.
 Example #3: Tryptophan (Trp) Operon

• Operon encodes 5 enzymes that catalyze synthesis

of tryptophan from chorismic acid
• If cell has enough tryptophan, then no need to
make more (that would waste energy)
• Presence of tryptophan should turn expression OFF
• Absence of tryptophan should turn expression ON
• Questions
o How is this different from the Lac system?
o Can you hypothesize a model for regulation?
 Trp Operon Regulation

• Repressor accounts for ~70 fold change in gene expression

• Attenuation accounts for ~10 fold change in gene expression
 Attenuation by TrpL
O

Tryptophan availability dictates rate of leader peptide synthesis

 Two Possible Base Pairing Patterns
• Sequence 2 can base pair with Sequence 3
• Alternatively, Sequence 1 can base pair with Sequence 2, while
Sequence 3 base pairs with Sequence 4
→ notice anything familiar about the 3:4 pair structure?
• Tryptophan availability dictates rate of leader peptide synthesis
• Given co-transcriptional translation, rate of protein synthesis dictates
pattern of RNA base pairing
 Tryptophan Starvation → Leader Translation Halted

• Synchronized co-transcriptional translation

• Low tryptophan → ribosome pauses at Trp codons in Sequence 1
• Transcription continues while translation pauses → Sequences 2
and 3 base pair, leaving Sequence 4 unpaired
• No terminator formation → transcription allowed to complete
Tryptophan Abundance→ Leader Translation Proceeds

• High tryptophan → no pausing at Trp codons in Sequence 1

• Ribosome moves along until it pauses at the stop codon and falls
off, allowing Sequence 1 to base pair with Sequence 2.
• Sequence 3 is transcribed, leading to formation of the 3:4 Rho-
independent transcription terminator hairpin
• RNA transcription aborts → no expression of structural genes
 Operon Regulation Review
Operon: lac ara trp
Regulate genes Regulate genes Regulate genes
Purpose: for catabolism for catabolism for synthesis of
of lactose of arabinose tryptophan
Negative
Yes Yes Yes
Control?

1. TrpR repressor
AraC repressor
LacI repressor binds to operator
binds to
How? binds to with co-repressor
operators and
operator trp
loops DNA
2. attenuation

Positive
Yes Yes No
Control?
CAP-cAMP CAP-cAMP
How? binds to CAP binds to CAP (N/A)
site site
Why Is Transcriptional Control Useful for Biotech?
• Biotechnological production frequently involves
using a host organism to generate products
encoded by plasmid DNA
• Maintaining plasmid stability
o Desired product may not be beneficial to host organism
o Continuous production drains energy → growth and survival
disadvantage → eventual loss of plasmid
o Controlling timing and duration of gene expression can increase
plasmid stability
• Production of toxic metabolites
o Desired product may be toxic to the host organism
o Allow cells to build up biomass without generating toxic product
o Induce product gene expression at controlled time point
 Promoters Commonly Used in Biotech
• Promoter characteristics
o Strength: how strongly a gene is expressed in the ON state
o Leakiness: how tightly repressed a promoter is in the OFF state
• PBAD is a weak, tight (i.e., non-leaky), all-or-none
promoter
• Wildtype (wt) lac promoter is weak → lacUV5 is a
stronger mutant of the lac promoter
• Trp promoter is leaky → unsuited for expression of toxic
genes
• Tac and trc are hybrid promoters
o Both contain -35 region from trp and -10 region from lac, with 16 (tac)
or 17 (trc) base pairs of separation
o Both repressed by lac repressor and induced by lactose or IPTG
o 3 times stronger than trp and 10 times stronger than lac in the
absence of the trp repressor
• Temperature-sensitive promoter
 pL Promoter from Bacteriophage λ
active
repressor
30°C protein

X
oL Target gene
pL
mRNA (repressible)
active
repressor
protein
42°C
cI gene unfolded, inactive
pcI
repressor protein
(constitutive)

oL Target gene
pL
 More Economically Viable Systems
• Inducer molecules can be prohibitively expensive
at large scale
• Changing the temperature of industrially sized
(>200 L) bioreactors is difficult
o Takes time to achieve temperature uniformity
o Energy consumption is costly

• Tryptone is a relatively cheap source of tryptophan

→ but need to overcome leakiness problem
• Can also use cell growth phase as an inducer-free
way of achieving inducible gene expression
 Dual-Plasmid Expression System
• Use ptrp to drive cI gene expression and pL to drive target gene expression
• Grow cells on cheap medium consisting of molasses and casein
hydrolysate (minimal free tryptophan)
• Add tryptone when it’s time to induce gene expression
• Incorporate pL regulatory component to reduce leakiness

No Tryptophan (ptrp de-repressed) With Tryptophan (ptrp repressed)

cI protein
synthesized No cI protein
synthesis
ptrp pL X ptrp pL
X
cI Gene cI Gene

Low-copy Low-copy
plasmid plasmid

No product gene Product gene

expression expression
 Stationary-Phase Expression System
• Bacterial cell growth goes through distinct phases
• Highest cell density at stationary phase
• Strategy: grow cells until stationary phase, then have
gene expression automatically kick in
• Use promoter recognized by stationary-phase σ factor σs
Fun with Transcriptional
Regulation
Repressilator
A circuit of 3 interlocking
repressors result in
oscillatory gene
expression in E. coli
M. B. Elowitz and S. Leibler. Nature. 403:335-
338(2000).

http://www.elowitz.caltech.edu/movies.html
 Logic Gates
• AND: “You must know the
basics and think creatively to
be a good scientist.”
• NOT: “You must not be late
or you won’t get any free
food.”
• OR: “You may apply for www.ee.surrey.ac.uk
college admission if you
have either received a high-
school diploma or passed
the GED test.”
• NOR: “To be eligible to vote,
you must be neither a minor
nor a convicted felon”
Quorum Sensing
• Vibrio fischeri bacteria colonize the light organ
of Hawaiian bobtail squids and help prevent the
squid from casting a shadow on moonlit nights
• Lighting only effective when bacteria reach a
certain density → quorum sensing

Light regulation in V. fisheri

 Edge Detectors

J.J. Tabor et al. Cell. 137:1272-1281(2009).

Circuit Layering

J.J. Tabor et al. Cell. 137:1272-1281(2009).

 Edge Detection of Complex Patterns

J.J. Tabor et al. Cell. 137:1272-1281(2009).

 Translational Regulation
• Ribosome binding sites (RBS) are essential to
translation initiation
• The Shine-Dalgarno (SD) sequence is an 8-nt RBS
with the 6-nt consensus sequence AGGAGG
• Mutations in the SD sequence alter ribosome
binding affinity and translation efficiency → can
regulate translation by using different RBS
sequences
• Physical blockage or sequestration of the RBS can
also affect translation efficiency