Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology 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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Part IV Regulation
Ch 16 Regulation in prokaryotes Ch 17
Regulation in eukaryotes Ch 18 Regulation during
development and in diseases (brief
introduction) Ch 19 Comparative genomics and
evolution of animal diversity (Not covered in the
lecture)
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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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• Chapter 16 Regulation principles and How genes
  are regulated in bacteria
• Chapter 17 Basic mechanism of gene expression in
  eukaryotes
• Chapter 18 The mechanism of RNAi and the role of
  miRNA in development and cancergenesis

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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 Examples of Gene Regulation after
  Transcription Initiation (Trp operon)
• TOPIC 4 The Case of Phage ? Layers of Regulation

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CHAPTER 16 Gene Regulation in Prokaryotes
Topic 1 Principles of Transcription Regulation
5/10/2005
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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. Gene expression is controlled at different
stages (?????????????)
Principles of Transcription Regulation

• The bulk of gene regulation takes place at the
  initiation of transcription.
• Some involve transcriptional elongation/terminatio
  n, RNA processing, and translation of the mRNA
  into protein.

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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

• RNAP binds many promoters weakly (?), activators
  that contain two binding sites to bind a DNA
  sequence and RNAP simultaneously can enhance the
  RNAP affinity with the promoters, and thus
  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.
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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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Principles of Transcription Regulation

• 5 Targeting promoter escape by some repressors

• Repressors can work in ways
• blocking the promoter binding.
• blocking the transition to the open complex.
• blocking promoter escape

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Some promoters are inefficient at more than one
step and can be activated by more than one
mechanism. Activation mechanisms include
recruitment (??) and allostery (??).
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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,
• integrate signals (some genes are activated when
  multiple signals are present).

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• 7. 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
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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 pathway whose expression is
  coordinately controlled.
• 2. Control elements, such as operator
  sequence.
• 3. Regulator gene(s) whose products recognize
  the control elements.

Sometimes are encoded by the gene under the
control of 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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1. Lactose operon contains a regulatory gene and
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 signal 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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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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The LAC operon
Fig 16-6
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The LAC operon
3. Lac repressor bound to the operator prevents
RNAP from binding to the promoter
The site bound by lac repressor is called the lac
operator (Olac ), and the Olac overlaps promoter
(Plac). Therefore repressor bound to the operator
physically prevents RNA polymerase from binding
to the promoter.
The LAC operon
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The LAC operon
Fig 16-8
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The LAC operon
4. CAP activates the Lac transcription through
recruitment of RNAP to the weak Plac
CAP has two binding sites, one interacts with the
CAP site on the DNA near promoter, and one
interacts with RNAP. This cooperative binding
ensures that RNAP effectively binds to Plac and
initiates transcription of LacZYA.
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 also interacts with the RNAP and recruit it
  to the promoter.

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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5. 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.
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.

Fig 16-13
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6 The activity of Lac repressor and CAP are
controlled allosterically by their signals.
Allolactose turn of Lac repressor cAMP 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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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
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7 Combinatorial Control (????) CAP controls
other genes as well

• 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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Regulation of Transcription Initiation in
Bacteria
Second example Alternative s factor
Alternative s factor (??s??) direct RNA
polymerase to alternative site of promoters
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? factor subunit bound to RNA polymerase for
transcription initiation (Ch 12)
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• Different ? factors binding to the same RNAP,
  conferring each of them a new promoter
  specificity.
• ?70 factors is 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 sfactors
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 and 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
• In the case of 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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• In the absence of NtrC and MerR, RNAP binds to
  the corresponding promoter to form a closed
  stable complex.
• NtrC activator induces a conformational change in
  the enzyme, triggering transition to the open
  complex
• MerR activator causes the allosteric effect on
  the DNA and triggers the transition to the open
  complex

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NtrC and MerR and allosteric activation
1. NtrC has ATPase activity and works from DNA
sites far 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 positio as a dimer ??NtrC (?)
interacts with ?54 (glnA promoter recognition) ??
NtrC ATPase activity provides energy needed to
induce a conformation change in polymerase??
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
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 Examples of Gene Regulation at Steps
After Transcription Initiation
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Examples of Gene Regulation at Steps After
Transcription Initiation
First example the tryptophan operon (??????)
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1. Amino acid biosynthetic operons are controlled
by premature transcription termination the trp
operon
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The TRP operon

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

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The TRP operon
The Trp repressor
(?????? )
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The TRP operon

1. Trp repressor is encoded by a separate operon
   trpR, and specifically interacts with the
   operator that overlaps with the promoter sequence
   
2. The repressor can only bind to the operator when
   it is complexed with tryptophan. Therefore, Try
   is a co-repressor and inhibits its own synthesis
   through end-product inhibition (negative
   feed-back regulation).

Remember the lac repressor acts as an inducer
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The TRP operon

• The repressor reduces transcription initiation by
  around 70-fold, which is much smaller than the
  binding of lac repressor.
• The repressor is a dimer of two subunits which
  has a structure with a central core and two
  flexible DNA-reading heads (carboxyl-terminal of
  each subunit )

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The TRP operon
trpR operon
trp operon
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The TRP operon
Attenuation (????) a regulation at the
transcription termination step a second
mechanism to confirm that little tryptophan is
available
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• Repressor serves as the primary switch to
  regulate the expression of genes in the trp
  operon
• Attenuation serves as the fine switch to
  determine if the genes need to be efficiently
  expressed

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Fig 16-19
Transcription of the trp operon is prematurally
stopped if the tryptophan level is not low
enough, which results in the production of a
leader RNA of 161 nt. (WHY?)
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1. Transcription and translation in bacteria are
   coupled (??????????????). Therefore, synthesis of
   the leader peptide immediately follows the
   transcription of leader RNA.
2. The leader peptide contains two tryptophan
   codons. If the tryptophan level is very low, the
   ribosome will pause at these sites.
3. Ribosome pause at these sites alter the secondary
   structure of the leader RNA, which eliminates the
   intrinsic terminator structure and allow the
   successful transcription of the trp operon.

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Fig 16-20 The leader RNA and leader peptide of
the trp operon
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High Trp
Complementary 34 termination of transcription
Low Trp
Complementary 23 Elongation of transcription
Fig 16-21
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Importance of attenuation

1. A typical negative feed-back regulation
2. Use of both repression and attenuation allows a
   fine tuning of the level of the intracellular
   tryptophan.
3. 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.
4. Provides an example of regulation without the use
   of a regulatory protein, but using RNA structure
   instead.

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Examples of Gene Regulation at Steps After
Transcription Initiation
Second example Riboswitches-a RNA structure
control mechanism
Riboswitches are regulatory RNA elements that act
as direct sensors of small molecule metabolites
to control gene transcription or translation.
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Box 4

1. Riboswitches operating at the level of
   transcription termination using an
   Antitermination mechanism.
2. Riboswitches operating at the level of
   translation, controlling the formation of an RNA
   structure that masks the ribosome binding site on
   mRNA.

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???
???
Tucker1 and Breaker, Current Opinion in
Structural Biology 2005, 15342348
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The 2nd structures of 7 riboswitches and
metabolites that they sense
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Examples of Gene Regulation at Steps After
Transcription Initiation
Third example Ribosomal proteins are
translational repressors of their own synthesis
a negative feedback
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• Challenges the ribosome protein synthesis
• 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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• Strategies to meet the challenges-Operon
• Organization of the ribosomal proteins to several
  operons (???) , each containing up to 11
  ribosomal protein genes
• 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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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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• Strategies to meet the challenges (cont)
• For each operon, one (or two) ribosomal proteins
  binds the mRNA near the translation initiation
  sequence, preventing the ribosome 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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7. 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 regulatory RNA in its
own mRNA.
Fig 16-23
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Key points of the chapter

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