Chapter 12 Gene Regulation in Prokaryotes

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Chapter 12Gene Regulation in Prokaryotes
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Gene Regulation Is Necessary?

• By switching genes off when they are not needed,
  cells can prevent resources from being wasted.
  There should be natural selection favoring the
  ability to switch genes on and off.
• Complex multicellular organisms are produced by
  cells that switch genes on and off during
  development.
• A typical human cell normally expresses about 3
  to 5 of its genes at any given time.
• Cancer results from genes that do not turn off
  properly. Cancer cells have lost their ability to
  regulate mitosis, resulting in uncontrolled cell
  division

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Classification of gene with respect to their
Expression

• Constitutive ( house keeping) genes
• Are expressed at a fixed rate, irrespective to
  the cell condition.
• They are essential for basic processes involving
  in cell replication and growth
• Controllable genes
• Are expressed only as needed. Their amount may
  increase or decrease with respect to their basal
  level in different condition.
• Their structure is relatively complicated with
  some response elements

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Regulation of gene expression

• lac operon was the first discovered example of a
  gene regulation system by Francois Jacob and
  Jacques Monod (Pasteur Institute, Paris, France)
• Studied the organization and control of the lac
  operon in E. coli.
• Earned Nobel Prize in Physiology / Medicine 1965.
  
• Studied 2 different types of mutations in the lac
  operon
• Mutations in protein-coding gene sequences.
• Mutations in regulatory sequences.

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The Principles of Transcription Regulation

• What are the regulatory proteins?
• Which steps of gene expression to be targeted?
• How to regulate? (recruitment, allostery,
  blocking, action at a distance, cooperative
  binding)

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1. Gene Expression is Controlled by Regulatory
Proteins (????)

• Gene expression is very often controlled by
  Extracellular Signals, which are communicated to
  genes by regulatory proteins
• Positive regulators or activators
  INCREASE the transcription
• Negative regulators or repressors
  
• DECREASE or ELIMINATE the transcription

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2. Most activators and repressors act at the
level of transcription initiation

• Why that?
• Transcription initiation is the most
  energetically efficient step to regulate. A wise
  decision at the beginning
• Regulation at this step is easier to do well than
  regulation of the translation initiation.

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• Regulation also occurs at all stages after
  transcription initiation. Why?
• Allows more inputs and multiple checkpoints.
• The regulation at later stages allow a quicker
  response.

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Promoter Binding (closed complex)
Promoter melting (open complex)
Promoter escape/Initial transcription
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Elongation
Termination
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3. Targeting promoter binding Many promoters
are regulated by activators (????) that help RNAP
bind DNA (recruitment) and by repressors (????)
that block the binding.
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• Generally, RNAP binds many promoters weakly. Why?
  
• Activators contain two binding sites to bind a
  DNA sequence and RNAP simultaneously, can
  therefore enhance the RNAP affinity with the
  promoters and increases gene transcription. This
  is called recruitment regulation (????).
• On the contrary, Repressors can bind to the
  operator inside of the promoter region, which
  prevents RNAP binding and the transcription of
  the target gene.

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a. Absence of Regulatory Proteins basal level
expression
b. Repressor binding to the operator
represses expression
c. Activator binding activates expression
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• 4 Targeting transition to the open complex
  Allostery regulation (????) after the RNA
  Polymerase Binding

In some cases, RNAP binds the promoters
efficiently, but no spontaneous isomerization
(???) occurs to lead to the open complex,
resulting in no or low transcription. Some
activators can bind to the closed complex,
inducing conformational change in either RNAP or
DNA promoter, which converts the closed complex
to open complex and thus promotes the
transcription. This is an example of allostery
regulation.
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Allostery regulation
Allostery is not only a mechanism of gene
activation , it is also often the way that
regulators are controlled by their specific
signals.
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• Repressors can work in ways
• blocking the promoter binding.
• blocking the transition to the open complex.

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• 5. Action at a Distance and DNA Looping. The
  regulator proteins can function even binding at a
  DNA site far away from the promoter region,
  through protein-protein interaction and DNA
  looping.

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DNA-binding protein can facilitate interaction
between DNA-binding proteins at a distance
Architectural protein
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6. Cooperative binding (recruitment) and
allostery have many roles in gene regulation

• For example group of regulators often bind DNA
  cooperatively (activators and/or repressors
  interact with each other and with the DNA,
  helping each other to bind near a gene they
  regulated)
• produce sensitive switches to rapidly turn on a
  gene expression. (11gt2)
• integrate signals (some genes are activated when
  multiple signals are present).

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• Topic 2 Regulation of Transcription Initiation
  
• Examples from Bacteria

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OPERON in gene regulation of prokaryotes

• Definition a cluster of genes in which
  expression is regulated by operator-repressor
  protein interactions, operator region, and the
  promoter.
• Its structure Each Operon is consisted of few
  structural genes( cistrons) and some cis-acting
  element such as promoter (P) and operator (O).
• Its regulation There are one or more regulatory
  gene outside of the Operon that produce
  trans-acting factors such as repressor or
  activators.
• Classification
• 1- Catabolic (inducible) such as
  Lac OPERON 2- Anabolic
  (repressible) such as ara OPERON
• 3- Other types

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General structure of an OPERON
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First example Lac operon
The lactose Operon (?????)
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Point 1 Composition of the Lac operon
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1. Lactose operon contains 3 structural genes and
2 control elements.
The enzymes encoded by lacZ, lacY, lacA are
required for the use of lactose as a carbon
source. These genes are only transcribed at a
high level when lactose is available as the sole
carbon source.
The LAC operon
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lacZ
codes for ß-galactosidase (?????) for lactose
hydrolysis
lacY

• encodes a cell membrane protein called lactose
  permease (???????) to transport Lactose across
  the cell wall

lacA
encodes a thiogalactoside transacetylase
(??????????)to get rid of the toxic
thiogalacosides
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The lacZ, lacY, lacA genes are transcribed
into a single lacZYA mRNA (polycistronic mRNA)
under the control of a single promoter Plac .
LacZYA transcription unit contains an operator
site Olac
position between bases -5 and 21 at the 3-end
of Plac
Binds with the lac repressor
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Control elements
21
-5
repressor
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Point 2 Regulatory proteins and their response
to extracellular signals
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2. An activator and a repressor together control
the Lac operon expression
The activator CAP (Catabolite Activator
Protein,????????) or CRP (cAMP Receptor
Protein,cAMP????) responses to the glucose
level. The repressor lac repressor that is
encoded by LacI gene responses to the
lactose. Sugar switch-off mechanism
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The LAC operon
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3. The activity of Lac repressor and CAP are
controlled allosterically by their signals.
Allolactose binding turn off Lac repressor cAMP
binding turn on CAP
Lactose is converted to allolactose by
b-galactosidase, therefore lactose can indirectly
turn off the repressor. Glucose lowers the
cellular cAMP level, therefore, glucose
indirectly turn off CAP.
The LAC operon
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Lac OPERON an inducible Operon
In the absence of lac
In the presence of lac
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CRP or CAP is positive regulator of Lac and some
other catabolic Operons
CRP Catabolic gene regulatory Protein CRP cAMP
receptor Protein CAP Catabolic gene Activating
Protein
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Regulation of lac Operon Expression
Off
Off
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Functional state of the E. coli lac operon in
the absence of lactose
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Functional state of the E. coli lac operon
growing on lactose
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Positive control of the lac operon with CAP
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Point 3 The mechanism of the binding of
regulatory proteins to their sites
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4. CAP and Lac repressor have opposing effects on
RNA polymerase binding to the promoter
Repressor binding physically prevents RNAP from
binding to the promoter, because the site bound
by lac repressor is called the lac operator (Olac
), and the Olac overlaps promoter (Plac).
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The LAC operon
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CAP binds to a site upstream of the promoter, and
helps RNA polymerase binds to the promoter by
physically interacting with RNAP. This
cooperative binding stabilizes the binding of
polymerase to Plac.
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Base pair sequence of controlling sites,
promoter, and operator for lac operon of E. coli.
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5. CAP interacts with the CTD domain of the
a-subunit of RNAP

• CAP interacts with the CTD domain of the
  a-subunit of RNAP and thus promotes the promoter
  binding by RNAP

a CTD C-terminal domain of the a subunit of RNAP
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Lactose/allolactose is a native inducer to
release RNA transcription from Plac. IPTG
(isopropyl-?-D-thiogalacto-pyranoside,???-ß-D-????
???? ), a synthetic inducer, can rapidly
stimulate transcription of the lac operon
structural genes. ? IPTG is used to induce the
expression of the cloned gene from lac promoter
in many vectors, such as pUC19.
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Gene X
No IPTG, little protein X With IPTG, a lot of
protein X
Back
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Second example The Trp operon of E. coli
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Trp OPERON a repressible example
In the absence of Trp
In the presence of Trp
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Regulation of the trp operon

• 1. Repressor/operator interaction
• When tryptophan is present, tryptophan binds to
  trpR gene product.
• trpR protein binds to the trp operator and can
  only bind to the operator, thus prevents
  transcription.
• Repression reduces transcription of the trp
  operon 70-fold.

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• 2. Molecular model for attenuation(????)
• Recall that a leader region (trpL) occurs between
  the operator and the trpE sequence.
• Within this leader is the attenuator sequence
  (att).
• att sequence contains a start codon, 2 Trp
  codons, a stop codon, and four regions of
  sequence that can form three alternative
  secondary structures.
• Secondary structure Signal
• Paired region 1-2 pause
• Paired region 2-3 anti-termination
• Paired region 3-4 termination

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Organization of the leader/attenuator trp operon
sequence.
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Attenuation model in Trp starved cells
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• Molecular model for attenuation (cont.)
• Position of the ribosome plays an important role
  in attenuation
• When Trp is scarce or in short supply (and
  required)
• Trp-tRNAs are unavailable, ribosome stalls at Trp
  codons and covers attenuator region 1.
• Region 1 cannot pair with region 2, instead
  region 2 pairs with region 3 when it is
  synthesized.
• Region 3 (now paired with region 2) is unable to
  pair with region 4 when it is synthesized.
• RNA polymerase continues transcribing region 4
  and beyond synthesizing a complete trp mRNA.

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Attenuation model in Trp non-starved cells
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• Molecular model for attenuation (cont.)
• Position of the ribosome plays an important role
  in attenuation
• When Trp is abundant (and not required)
• Ribosome does not stall at the Trp codons and
  continues translating the leader polypeptide,
  ending in region2.
• Region 2 cannot pair with region 3, instead
  region 3 pairs with region 4.
• Pairing of region 3 and 4 is the attenuator
  sequence and acts as a termination signal.
• Transcription terminates before the trp
  synthesizing genes are reached.

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The attenuators of some operons
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