Gene Regulation

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• Molecular Biology Course

• Chapter 16
• Gene Regulation
• in Prokaryotes

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outline
There are four major parts in this chapter
Principles of Transcriptional Regulation
Regulation of Transcription Initiation
Examples of Gene Regulation at Steps after
Transcription Initiation The Case of
Phage?Layers of Regulation
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Part One
CHAPTER 16 Gene Regulation in Prokaryotes

• Principles of Transcriptional
• Regulation

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• Gene Expression is Controlled by Regulation
  Proteins Activators and Repressors
• 1.Activators, or Positive regulators, increase
  transcription of the regulated gene
• Repressors, or negative regulators, decrease
  or eliminate that transcription.
• 2. Many Promoters Are Regulated by Activation
  that Help RNA Polymerase Bind DNA and by
  Repressors that Block that Binding.

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Fig 16-1
a. Absence of Regulatory Proteins (operator)
b. To Repress Expression
c. To Activate Expression
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3.Some Activators Work by Allostery and Regulate
Steps after RNA Polymerase Binding In some
cases, RNA Polymerase binds efficiently unaided
and forms a stable closed complex, which does
not spontaneously undergo transition to the open
complex. Activator that stimulate this kind of
promoter wrk by triggering a conformational
change in either RNA Polymerase or DNA. That is,
they interact with the stable closed complex and
induce a conformational change that causes
transition to the open complex.
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This mechanism is an example of allostery.
Fig 16-2
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• Action at a Distance and DNA Looping.

• Some proteins interact with each other even when
  bound to sites well separated on the DNA

Fig 16-3
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DNA-bending protein can facilitate interaction
between DNA-binding proteins at a distance
Fig 16-4
In this example, we also call the DNA-binding
protein architectural proteins.
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• Cooperative Binding and Allostery have Many Roles
  in Gene Regulation
• Cooperative binding the activator interacts
  simultaneously with DNA and polymerase and so
  recruits the enzyme to the promoter.
• Two roles IN RESPONSE TO SMALL CHANGES
  SENSITIVELY and SERVE TO INTEGRATE SIGNALS
• 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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• Antitermination and Beyond Not All of Gene
  Regulation Targets Transcription Initiation
• The bulk of gene regulation takes place at the
  initiation of transcription in both eukaryotes
  and bacteria.
• But regulation is certainly not restricted to
  that step in either class of organism. In this
  chapter we will see examples, in bacteria, of
  gene regulation that involve transcriptional
  elongation, RNA processing, and translation of
  the mRNA into protein.

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Part Two
CHAPTER 16 Gene Regulation in Prokaryotes
Regulation of Transcription Initiation Examples
From Bacteria
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EXAMPLE ONE------LAC OPERON
The lactose (Lac) Operon (?????)
Fig 16-5
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Lactose operon a regulatory gene and 3
stuctural genes, and 2 control elements
Regulatory gene
Structural Genes
Cis-acting elements
DNA
lacI
lacZ
lacY
lacA
PlacI
Olac
Plac
m-RNA
Protein
ß -Galactosidase
Transacetylase
Permease
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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• An Activator and a Repressor Together Control the
  lac Genes

The activator is called CAP( Catabolite Activator
Protein ) .CAP can bind DNA and activate the lac
genes only in the absence of glucose. The lac
repressor can bind DNA and repress transcrition
only in the absence of lactose. Both CAP and lac
repressor are DNA-binding proteins and each binds
to a specific site n DNA at or near the lac
promoter.
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The LAC operon
Fig 16-6
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• CAP and lac repressor have opposing effects on
  RNA polymerase binding to the lac promoter

1.Lac operator ------the site bound by lac
repressor This 21 bp sequence is twofold
summetric and is recognized by two subunits of
lac repressor, one binding to each half-site.
Fig 16-7
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lac operator overlaps promoter, and so repressor
bound to the operator physically prevents RNA
polymerase from binding to the promoter.
Fig 16-8
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2. CAP
CAP binds as a dimer to a site similar in length
to the lac operator, but different in sequence
and location. CAP has separate activating and
DNA-binding surfaces.
Fig 16-9
At the promoter,where there is no UP-element, a
CTD binds to CAP and adjacent DNA instead.
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CAP and lac repressor bind DNA using a common
structural motif

• 1.The Same
• A. The protein binds as a homodimer to a site
  that is an inverted repeat or near repeat.
• B.Both CAP and lac repressor bind DNA using a
  helix-turn-helix motif.

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• One of the two ?helices in helix-turn-helix
  domain is the recognition helix that can fits
  into the major groove of the DNA.

Fig 16-11
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• The second helix of the helix-turn-helix domain
  sits across the major groove an makes contact
  with the DNA backbone , ensuring proper
  presentation of the recognition helix, and at
  the same time adding binding energy to the
  overall protein-DNA interaction.

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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
The LAC operon
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2. The Difference

• Lac repressor binds as a tetramer, with each
  operator is contacted by a repressor dimer.

Fig 16-13
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• In some cases, other regions of the protein,
  outside the helix-turn-helix domain, also
  interact with the DNA.
• In many cases, binding of the protein does not
  alter the structure of the DNA .In some cases,
  however, various distortions are seen in the
  protein-DNA complex.

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• The activity of Lac repressor and CAP are
  controlled allosterically by their signals

Binding of the corresponding signals alter the
structure of these two regulatory proteins
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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 .
Lactose is present, the low basal level of
permease allows its uptake, andß-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
The LAC operon
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• Combinatorial Control CAP controls other genes
  as well

• The lac genes provide an example of signal
  integration their expression is controlled by
  two signals, each of which is communicated to the
  genes via a single regulatorthe lac repressor
  and CAP, respectively.

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• A regulator (CAP) works together with different
  repressor 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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• Combinatorial control is a characteristic feature
  of gene regulation. More complex organismshigher
  eukaryotes in particular---tend to have more
  signal integration.

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EXAMPLE TWO---- ALTERNATIVE sFACTORS
Alternative s factor direct RNA polymerase to
alternative site of promoters
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Recall from Chapter 12 that it is the ssubunit of
RNA polymerase that recognizes the promoter
suquences.
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Promoter recognition
Different sfactors binding to the same RNA Pol
Confer each of them a new promoter specificity
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Many bacteria produce alternative sets of
sfactors to meet the regulation requirements of
transcription under normal and extreme growth
condition.
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• Heat shock--- ?32
• When E.coliis subject to heat shock, the amount
  of this new sfactor increases in the cell, it
  displaces s70 from a proportion of RNA
  polymerases ,and directs those enzymes to
  transcribe genes whose products protect the cell
  from the effects of heat shock. The level of ?32
  is increased by two mechanisms first, its
  translation is stimulated---that is,its mRNA is
  translated with greater efficiency after heat
  shock than it was before and second, the protein
  is transiently stabilized.

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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 .
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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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Normal bacterial holoenzyme
Express early genes
Encode s28
Express middle genes (gene 34 and 33 )
Encodesfactor for transcription of late genes
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EXAMPLE THREE---NtrC and MerR

• NtrC and Mert Transcriptional Activators that
  Work by Allostery Rather than by Recruitment

NtrC controls expression of genes involved in
nitrogen metabolism, such as the glnA gene. At
the glnA gene, Ntrc induces a conformational
change in the RNA Polymerase, triggering
tansition to the open complex.
MerR controls a gene called merT. Like NtrC, MerR
induces a conformational change in the inactive
RNA polymerase-promoter complex, and this change
can trigger open complex formation.
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• NtrC Has ATPase Activity and Works from DNA Sites
  Far from the Gene

• NtrC has separate activating and DNA-binding
  domains, and binds DNA only when the nitrogen
  levels are low.

Fig 16-15 activation by NtrC
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The major process
Low nitrogen levels
Trigger polymerase to initiate transcription
NtrB phosphorylates NtrC
ATP hydrolysis and conformation change in
polymerase
NtrCs DNA-binding domain revealed
NtrC binds four sites located some 150 base pairs
upstream of the promoter
NtrC interacts with ?54
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• 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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The merT promoter is unusual. The distance
between the -10 and -35 elements is 19bp instead
of the 15 to 17 bp typically found in an
eddicient ?70 promoter. So, these two elements
recognized by ? are neither optimally seperated
nor aligned.
Fig 16-15 a
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The binding of MerR locks the promoter in the
unpropitious conformation in the absence of Hg2 .
Fig 16-15 b
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When Hg2 is present, MerR binds Hg2 and undergo
conformational change, which twists the promoter
to restore it to the structure close to a strong
?70 promoter
Just like this
Fig 16-15 c
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In this new configuration , RNA polymerase can
efficiently initiate transcription.
Fig 16-15
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• Some repressors hold RNA polymerase at the
  promoter rather than excluding it

• Repressors work in different ways
• By binding to a site overlapping the promoter, it
  blocks RNA polymerase binding. (lac repressor)
• The protein holds the promoter in a conformation
  incompatible with tanscription initiation.(the
  MerR case)
• 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)

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EXAMPLE FOUR----araBAD OPERON

• AraC and control of the araBAD operon by
  antiactivation

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

Fig 16-18
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The magnitude of induction of the araBAD promoter
by arabinose is very large , and for this reason
the promoter is often used in expression
vectors. Expression vectors are DNA constructs in
which efficient synthesis of any protein can be
ensured by fusing a gene to a strong promoter .
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CHAPTER 16 Gene Regulation in Prokaryotes
Part Three Examples of
Gene Regulation at Steps After
Transcription Initiation
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• Amino acid biosynthetic operons are controlled by
  premature transcription termination

the tryptophan operon
Fig 16-19
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• The trp operon encodes five structural genes
  required for tryptophan synthesis.
• These genes are regulated to efficiently express
  only when tryptophan is limiting.
• There are two layers of regulation involved
  (1) transcription repression by the Trp
  repressor (initiation) (2) attenuation

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The Trp repressor ---the first layer
of regulation When tryptophan is present, it
binds the Trp repressor and induces a
conformational change in that protein, enabling
it to bind the trp operator and prevent
transcription. When the tryptophan concentration
is low, the Trp repressor is free of its
corepressor and vacates its operator , allowing
the synthesis of trp mRNA to commence from the
adjacent promoter. The ligand that controls the
activity of the trp repressor acts not as an
inducer but as a corepressor.
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Attenuation ---the
second layer of regulation
The key to understanding attenuation came from
examining the suquence of the 5 end of trp
operon mRNA. 161 nucleotides of RNA are made from
tryptophan promoter before RNA polymerase
encounters the first codon of trpE. Near the end
of this leader sequence ,and before trpE , is a
transcription terminator, composed of a
characteristic hairpin loop in the RNA.
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The hairpin loop is followed by 8 uridine
residues. At this so-called attenuator ,
transcription usually stops,yielding a leader RNA
139 nucleotides long.
Fig 16-20
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• Thre features of the leader sequence
• There is a second hairpin (besides the
  terminator hairpin) that can form between regions
  1 and 2 of the leader sequence.
• region 2 also is complementary to region 3 thus
  , yet another hairpin consisting of regions 2 and
  3 can form and when it does prevent the
  terminator hairpin (3,4) from forming.
• The leader RNA contains an open-reading frame
  encoding a short leader peptide of 14 amino
  acids, and this open-reading frame is preceded by
  a strong ribosome binding site.

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The sequence encoding the leader peptide has a
striking feature two tyrptophan codons in a
row. The fuction of these codons is to stop a
ribosome attempting to translate the leader
peotide.
Above all , how transcription termination at the
trp operon attenuator is controlled by the
availability of tryptophan
?
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Fig 16-21
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The Importance of Attenuation

• Use of both repression and attenuation allows a
  fine tuning of the level of the intracellular
  tryptophan.
• Attenuation alone can provide robust regulation
  other amino acids operons like his and leu have
  no repressors and rely entirely on attenuation
  for their regulation.
• Provides an example of regulation without the use
  of a regulatory protein, but using RNA structure
  instead.
• A typical negative feed-back regulation.

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• Ribosomal Protein Are Translational Repressors of
  their Own Synthesis

• The ribosome protein synthesis has challenges
• Each ribosome contains some 50 distinct proteins
  that must be made at the same rate
• The rate of the ribosome protein synthesis is
  tightly closed to the cells growth rate

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How to overcome the challenges

• Control of ribosome protein genes is simplified
  by their organization to several operons , each
  containing genes for up to 11 ribisomal proteins.
• Some nonribosomal proteins whose synthesis is
  also linked to growth rate are contained in these
  operons, including those for RNAP subunits a, b
  and b.
• The primary control is at the level of
  translation, not transcription.

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How to overcome the challenges

• For each operon,one ribosomal protein binds the
  messenger near the translation initiation
  sequence of the first genes in the operon,
  preventing ribosomes from binding and initiating
  translation.
• Repressing translation of the first gene also
  prevents expression of some or all of the rest.
• The strategy is very sensitive. A few unused
  molecule of protein L4, for example, will shut
  down synthesis of that protein and other proteins
  in this operon.

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Ribosomal protein operons
The protein that acts as a translational
repressor of the other proteins is shaded red.
Fig 16-22
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The mechanism of one ribosomal protein also
functions as a regulator of its own translation
the protein binds to the similar sites on the
ribosomal RNA and to the regulated mRNA

Fig 16-23
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Part Four
CHAPTER 16 Gene Regulation in Prokaryotes
The Case of Phage ? Layers of Regulation
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Bacteriophage ?is a virus that infects E.coli.
Upon infection, the phage can propagate in either
of two ways lytically or lysogenically.
A lysogen is extremely stable under normal
circumstances ,but the phage dormant within
it---the prophage---can efficiently switch to
lytic growth if the cell is exposed to agents
that damaged DNA . This switch from lysogenic to
lytic growth is called lysogenic induction.
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Lytic cycle and Establishment of lysogeny
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• Alternative Patterns of Genes Expression Control
  Lytic and Lysogenic Growth

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• Regulatory Proteins and Their Binding Sites

The cI gene encodes ?repressor, a protein of two
domains joined by a flexible linker egion.
?repressor can both activate and repress
trandcription. Cro only represses transcription.
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• Repressor and Cro Bind in Different Patterns to
  Control Lytic and Lysogenic Growth

Repressor bound to OR1and OR2 turns off
transcription from PR . And Repressor bound at
OR2 contacts RNA polymerase at PRM, activating
expression of the cI gene. OR3 lies with PRM Cro
bound there represses transcription of cI.
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• Another Activator, ?cII, Controls the Decision
  between Lytic and Lysogenic Growth upon Infection
  of a new Host

cII is a transcriptional activator. It binds to a
site upstream of a promoter called PRE and
stimulates transcription of the cI gene from that
promoter.
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• Transcriptional Antitermination in ? Development

The transcripts controlled by ?N and Q proteins
are initiated perfectly well in the absence of
those regulators. But the transcripts terminate a
few hundred to a thousand nucleotides downstream
of the promoter unless RNA polymerase has been
modified by the regulator ?N and Q protein are
therefore called antiterminators.
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Key points of the chapter

• Principles of gene regulation. (1) The targeted
  gene expression events (2) the mechanisms by
  recruitment/exclusion or allostery
• Regulation of transcription initiation in
  bacteria the lac operon, alternative s factors,
  NtrC, MerR, Gal rep, araBAD operon
• Examples of gene regulation after transcription
  initiation the trp operon, riboswitch,
  regulation of the synthesis of ribosomal proteins