Welcome to MB Class

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Welcome to MB Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
2005-5-10
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The revised central dogma
The structure of DNA and RNA
??????
??????
RNA processing
Gene regulation
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Part IV Regulation
Ch 16 Transcriptional regulation in
prokaryotes Ch 17 Transcriptional regulation in
eukaryotes Ch18 Regulatory RNAs Ch 19 Gene
regulation in development and evolution Ch 20
Genome Analysis and Systems Biology
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Expression of many genes in cells are regulated
Housekeeping genes expressed constitutively,
essential for basic processes involving in cell
replication and growth. Inducible genes
expressed only when they are activated by
inducers or cellular factors.
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Surfing the contents of Part IV --The heart of
the frontier biological disciplines
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Some of the peoples who significantly contribute
to the knowledge of gene regulation
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• Molecular Biology Course

• Chapter 16
• Gene Regulation
• in Prokaryotes

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• TOPIC 1 Principles of Transcriptional Regulation
  watch the animation
• TOPIC 2 Regulation of Transcription Initiation
  Examples from Bacteria (Lac operon, alternative s
  factors, NtrC,MerR, Gal rep, araBAD operon)
• TOPIC 3 The Case of Phage ? Layers of Regulation

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CHAPTER 16 Gene Regulation in Prokaryotes
Topic 1 Principles of Transcription Regulation

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

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1. Gene Expression is Controlled by Regulatory
Proteins (????)
Principles of Transcription Regulation

• 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
Principles of Transcription Regulation

• 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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Fig 12-3-initiation
Promoter Binding (closed complex)
Promoter melting (open complex)
Promoter escape/Initial transcription
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Fig 12-3-Elongation and termination
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.
Principles of Transcription Regulation
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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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Fig 16-1
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

Principles of Transcription Regulation
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
Fig 16-2
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.
• blocking promoter escape

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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.

Principles of Transcription Regulation
Fig 16-3
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Fig 16-4 DNA-binding protein can facilitate
interaction between DNA-binding proteins at a
distance
Fig 16-4
Architectural protein
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6. Cooperative binding (recruitment) and
allostery have many roles in gene regulation
Principles of Transcription 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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Watch the animation-regulation of the
transcription initiation!
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CHAPTER 16 Gene Regulation in Prokaryotes
Topic 2 Regulation of Transcription Initiation
Examples from Bacteria
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• Operon a unit of prokarytoic gene expression and
  regulation which typically includes
• 1. Structural genes for enzymes in a specific
  biosynthetic and metabolic pathway whose
  expression is coordinately controlled.
• 2. Control elements, such as operator
  sequence.
• 3. Regulator gene(s) whose products recognize
  the control elements. These genes is usually
  transcribed from a different promoter.

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Control element
Structural genes
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Regulation of Transcription Initiation in
Bacteria
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.
Fig 16-5
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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codes for ß-galactosidase (?????) for lactose
hydrolysis
lacZ
encodes a cell membrane protein called lactose
permease (???????) to transport Lactose across
the cell wall
lacY
encodes a thiogalactoside transacetylase
(??????????)to get rid of the toxic
thiogalacosides
lacA
The LAC operon
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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
The LAC operon
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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
The LAC operon
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3. The activity of Lac repressor and CAP are
controlled allosterically by their signals.
Allolactose binding turn of 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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The LAC operon
Fig 16-6
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Response to lactose
Lack of inducer the lac repressor block all but
a very low level of trans-cription of lacZYA .
When Lactose is present, the low basal level of
permease allows its uptake, and b-galactosidase
catalyzes the conversion of some lactose to
allolactose. Allolactose acts as an inducer,
binding to the lac repressor and inactivate
it.
Presence of lactose
i
p
o
z
y
a
Inactive
Permease
Transacetylase
b-Galactosidase
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Response to glucose ?????CRP??????
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Point 3 The mechanism of the binding of
regulatory proteins to their sites
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The LAC operon
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).
The LAC operon
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The LAC operon
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.
The LAC operon
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The LAC operon
Fig 16-8
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The LAC operon
5. CAP interacts with the CTD domain of the
a-subunit of RNAP
The LAC operon
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• CAP site has the similar structure as the
  operator, which is 60 bp upstream of the start
  site of transcription.
• CAP interacts with the CTD domain of the
  a-subunit of RNAP and thus promotes the promoter
  binding by RNAP.

Fig 16-9
a CTD C-terminal domain of the a subunit of RNAP
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The LAC operon
CAP binds as a dimer
a CTD
Fig 16-10. CAP has separate activating and
DNA-binding surface
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6. CAP and Lac repressor bind DNA using a common
structural motif helix-turn-helix motif
Fig 16-11
One is the recognition helix that can fits into
the major groove of the DNA. Another one sits
across the major grove and makes contact with the
DNA backbone.
The LAC operon
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• DNA binding by a helix-turn-helix motif

Fig 16-12 Hydrogen Bonds between l repressor and
the major groove of the operator.
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• Lac operon contains three operators the primary
  operator and two other operators located 400 bp
  downstream and 90 bp upstream.
• Lac repressor binds as a tetramer (???), with
  each operator is contacted by a repressor dimer
  (???). respectively.

DNA looping
Fig 16-13
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7 Combinatorial Control (????) CAP controls
other genes as well.

• A regulator (CAP) works together with different
  repressors at different genes, this is an example
  of Combinatorial Control.
• In fact, CAP acts at more than 100 genes in
  E.coli, working with an array of partners.

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Regulation of Transcription Initiation in
Bacteria
Second example Alternative s factor
Alternative s factors (??s??) direct RNA
polymerase to alternative promoters.
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? factor subunit bound to RNA polymerase for
transcription initiation (Ch 12)
Fig 12-7 s and a subunits recruit RNA pol core
enzyme to the promoter
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• Different ? factors binding to the same RNAP,
  conferring each of them a new promoter
  specificity.
• ?70 factors is the most common one in E. coli
  under the normal growth condition.

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Many bacteria produce alternative sets of
sfactors to meet the regulation requirements of
transcription under normal and extreme growth
condition. Bacteriophage has its own sfactors
E. coli Heat shock ?32
Bacteriophage s factors
Sporulation in Bacillus subtilis
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Heat shock (???)

• Around 17 proteins are specifically expressed in
  E. coli when the temperature is increased above
  37ºC.
• These proteins are expressed through
  transcription by RNA polymerase using an
  alternative ? factor ?32 coded by rhoH gene. ?32
  has its own specific promoter consensus sequences.

Alternative s factors
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Bacteriophages
Many bacteriophages synthesize their own
sfactors to endow the host RNA polymerase with a
different promoter specificity and hence to
selectively express their own phage genes .
Alternative s factors
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Alternative s factors
Fig 16-14
B. subtilis SPO1 phage expresses a cascade of
sfactors which allow a defined sequence of
expression of different phage genes.
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Regulation of Transcription Initiation in
Bacteria
Third example NtrC and MerR use allosteric
activation
Transcriptional activators NtrC and MerR work by
allostery rather than by recruitment.
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• Review
• The majority of activators work by recruitment,
  such as CAP. These activators simply bring an
  active form of RNA polymerase to the promoter.
• The beautiful exceptions allosteric activation
  by NtrC and MerR.
• In allosteric activation RNAP initially binds
  the promoter in an inactive complex, and the
  activator triggers an allosteric change in that
  complex to activate transcription.

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NtrC and MerR and allosteric activation
1. NtrC has ATPase activity and works at DNA
sites far away from the gene.

• NtrC controls expression of genes involved in
  nitrogen metabolism (???), such as the glnA gene.
• NtrC has separate activating and DNA-binding
  domains, and binds DNA only when the nitrogen
  levels are low.

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Low nitrogen levels (????)??NtrC phosphorylation
and conformational change?? NtrC (?) binds DNA
sites at -150 bp position as a dimer (?)??NtrC
interacts ?54 in RNAP bound to the glnA promoter
?? NtrC ATPase activity provides energy needed to
induce a conformation change in RNAP??
transcription STARTs
Fig 16-15 activation by NtrC
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NtrC and MerR and allosteric activation
2. MerR activates transcription by twisting
promoter DNA

• MerR controls a gene called merT, which encodes
  an enzyme that makes cells resistant to the toxic
  effects of mercury (???)
• In the presence of mercury (?), MerR binds to a
  sequence between 10 and 35 regions of the merT
  promoter and activates merT expression.

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As a ?70 promoter, merT contains 19 bp between
10 and 35 elements (the typical length is 15-17
bp), leaving these two elements recognized by ?70
neither optimally separated nor aligned.
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Fig 16-15 Structure of a merT-like promoter
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When Hg2 is absent, MerR binds to the promoter
and locks it in the unfavorable conformation When
Hg2 is present, MerR binds Hg2 and undergoes
conformational change, which twists the promoter
to restore it to the structure close to a strong
?70 promoter
Fig 16-15
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• Repressors work in many ways-review
• Blocking RNA polymerase binding through binding
  to a site overlapping the promoter. Lac repressor
• Blocking the transition from the closed to open
  complex. Repressors bind to sites beside a
  promoter, interact with polymerase bound at that
  promoter and inhibit initiation. E.coli Gal
  repressor
• Blocking the promoter escape. P4 protein
  interaction with PA2c (bacteriophage f29 )

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Regulation of Transcription Initiation in
Bacteria
Fourth example araBAD operon
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The araBAD operon
1. AraC and control of the araBAD operon by
anti-activation

• The promoter of the araBAD operon from E. coli is
  activated in the presence of arabinose (????) and
  the absence of glucose and directs expression of
  genes encoding enzymes required for arabinose
  metabolism. This is very similar to the Lac
  operon.

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• Different from the Lac operon, two activators
  AraC and CAP work together to activate the araBAD
  operon expression

194 bp
CAP site
DNA looping
Fig 16-18
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• Because the magnitude of induction of the araBAD
  promoter by arabinose is very large, the promoter
  is often used in expression vector.
• If fusing a gene to the araBAD promoter, the
  expression of the gene can be easily controlled
  by addition of arabinose(????).
• What is an expression vector ? The answer is in
  the Methods part.

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CHAPTER 16 Gene Regulation in Prokaryotes
Topic 3 The Case of Bacteriophage l Layers of
Regulation
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• Bacteriophage l is a virus that infects E.
  coli. Upon infection, the phage can propagate in
  either of two ways lytically or lysogenically.

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The phage l has a 50-kb genome and 50 genes.
Most of these genes encode protein for
replication, packing or lysis.
How the lytic and lysogenic growth is regulated?
---regulatory proteins and cis-acting control
elements.
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1. Alternative patterns of gene expression control
   lytic and lysogenic growth.

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• Fig. 16-21 Promoters in the right and left
  control regions of phage l

Fig. 16-22 Transcription in the l control
regions in lytic and lysogenic growth
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2. Regulatory Proteins and Their Binding Sites
The cI gene encodes l repressor, that can both
activate and repress transcription
As a repressor similarly as Lac repressor
(?) As an activator similarly as CAP (?)
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• l repressor
• As a repressor, it binds to sites that
• overlap the promoter and excludes RNA
• polymerase
• As an activator, it works like CAP by
• recruitment.

Cro (another regulatory protein), stands for
control of repressor and other things. It is a
single domain protein that binds as a dimer to
17-bp DNA sequences using a HTH motif. It only
represses transcription.
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There are 6 operators in the right (3) and left
(3) control regions of bacteriophage l.
Sequences are not identical

• repressor and Cro can each bind to any one of
  six operators, but with dramatically different
  affinity.
• repressor binds OR1 most easily while Cro binds
  OR3 with highest affinity.
• l repressor binds OR1 tenfold better than OR2.
  Cro binds OR3 tenfold better than OR1 and OR2.

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3. l repressor binds to operator sites
cooperatively. Two dimmers of repressor bind
cooperatively to OR1 and OR2, The binding at OR1
helps the binding at OR2.
Monomer
Dimer
Tetramer
10-fold low affinity
High affinity
Not bound
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• Box 16-3 Concentration, affinity, and cooperative
  binding

Two factors determine whether two interacting
molecules find and bind each others (1) the
binding affinity (2) their concentrations.
The curve of l repressor binding to its operator
DNA.
Binding of a protein to a single site
Cooperativity binding can be expressed in terms
of increased affinity.
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The benefit of cooperative binding of regulatory
proteins is to ensure dramatic changes in the
expression level of a given gene even in response
to small changes in the level of the control
signal.
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4. Repressor and Cro bind in different patterns
to control lytic and lysogenic growth
Lysogen
Lytic growth
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5. Lysogenic induction requires proteolytic
cleavage of l repressor

1. DNA damage activates RecA in E. coli
2. RecA stimulates l repressor to undergo
   autocleavage, resulting in the removal the
   C-terminal domain and the immediate loss of
   dimerization and binding cooperativity.
3. Repressor dissociates from OR1-OR2 OR1-OR2,
   which triggers transcription from PR and PL
4. leading to lytic growth.

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• For induction to work efficiently, the level of
  repressor in a lysogen must be tightly regulated.
  How?
• Keep it not too low by positive autoregulation l
  repressor binding at OR2 activates its own
  transcription from PRM.
• Keep it not too high by negative autoregulation
  when the repressor level goes too high, it will
  bind to OR3 as well, which will prevents
  transcription from PRM.

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6. Negative autoregulation of repressor requires
long-distance interactions and a large DNA loop
cooperative binding at OR3 and OL3.
Fig. 16-27

• When repressor level is high, it occupies both
  OR1-OR2 and OL1-OL2, and the interaction between
  two tetramer forms the repressor octomer and
  bring together OR3 and OL3 for another
  cooperative binding of the repressor.

Thats why lysogeny can be so stable while also
ensuring that induction is very efficient.
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• Figure 16-28 Interactions between the c-terminal
  domain of repressors.

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7. Another activator, l CII, controls the
decision between lytic and lysogenic growth upon
infection of a new host-an earlier event.

• cII is transcribed from PR and cIII is
  transcribed from PL.
• CII protein is a transcriptional activator that
  binds to PRE and stimulate the transcription of
  cI gene (l repressor).

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• Establishment of lysogeny synthesis of the
  essential lysogenic l repressor is established by
  transcription from one promoter and then
  maintained by transcription from another one.

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• Establishment of lysogeny-a bigger view
• PR and PL is constitutive promoters that promote
  transcription once the phage enter the cells.
• PR directs the synthesis of both Cro and CII
  proteins. Cro favors lytic development while CII
  favors lysogentic growth by activating the
  synthesis of l repressor.
• The efficiency with which CII directs
  transcription of cI gene (l repressor) is
  critical in deciding the lysogeny.
• ??? What determines CII efficiency?

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8. The number of phage particles infecting a
given cell affects whether the infection proceeds
lytically or lysogenically. When more CII
proteins are made from more infected phages,
there is a larger chance to produce enough l
repressor to produce lysogeny. 9. Growth
conditions of E. coli control the stability of
CII protein and thus the lytic/lysogenic choice.
Infection of Healthy and growing vigorously
bacterial cells gtgtCII is unstable gtgtpropagates
lytically because. When conditions are poor for
bacterial growth gtgtCII becomes stable gtgt form
lysogens and sit tight. CII is degraded by a
specific protease FtsH
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10. Transcriptional Antitermination in l
development examples of regulation after
transcription initiation.
Two l phage regulatory proteins N and Q, called
antiterminators, prevent the termination at some
termination sites and promotes the transcription
of the early late and late genes for the lytic
growth of the phage.
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Gene expression in l lytic growth

• Three phases
• Immediate early Transcription starts at PR and
  PL that flank the cI and stops at the r-dependent
  terminators (t) after the N and cro genes
• Delayed early Transcription begins at the same
  promoters, but bypasses the terminators by virtue
  of the N gene product, N, which is an
  antiterminator
• Late Transcription begins at a new promoter PR
  it would stop short at the t without the Q gene
  product, Q, another antiterminator.

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N proteins binds to the RNA
Q proteins bind to the QBE DNA site.
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Antitermination by N protein

• The gene surrounding N are depicted along with
  the leftward promoter (PL) and operator (OL), the
  terminator and the nut site.
• Transcription in the absence of N
• Transcription in the presence of N

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Key points of the chapter

1. Principles of gene regulation. (1) who regulate?
   (2) where to target? (3) How to regulate?
2. Regulation of transcription initiation in
   bacteria the lac operon, alternative s factors,
   NtrC, MerR, araBAD operon.
3. The case of l phage--layers of regulation l
   repressor and Cro and their binding control of
   the lytic and lysogenic growth lysogenic
   induction control of the decision to lytic or
   lysogenic growth by l CII Antiterminators.