Molecular biology of the gene Part 4: regulation

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Molecular biology of the genePart 4 regulation
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Chapter16 Gene Regulation in Prokaryotes
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• Topic1 principles of transcriptional regulation.
• Topic2 regulation of transcription initiation
  examples from bacteria.
• Topic3 examples of gene regulation at steps
  after transcription initiation.
• Topic4 the case of phage ? layers of
  regulation.

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Out Line

• 1.Principles of Transcriptional Regulation
• 2.Regulation of Transcription Initiation
  Examples from Bacteria
• 3.Examples of Gene Regulation at Steps after
  Transcription Initiation
• 4.The Case of Phage ?Layers of Regulation

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• The regulation expression---mechanisms that
  increase or decrease expression of a given gene
  as the requirement its product varies.
• The bulk of this chapter focuses on the
  regulation of transcription initiation in
  bacteria, also consider mechanism of gene
  regulation that operate at steps after
  transcription initiation ,including transcription
  antitermination and translation.

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Topic 1 Principle of transcriptional regulation
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Gene expression is controlled by regulatory
proteins

• In bacteria , signals are communicated to genes
  by regulatory proteins, which come in two types
  positive regulators, or activators and negative
  regulators, or repressors.

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Many promoters are regulated by activators that
help RNA polymerase bind DNA and by repressors
that block that binding
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• In the absence of both activator and repressor ,
  RNA polymerase occasionally binds the promoter
  spontaneously and initiates a low level (basal
  level) of transcription.
• The repressor blocks polymerase binding to the
  promoter. The site on DNA where a repressor binds
  is called an operator.
• An activator helps polymerase bind the promoter.
  this mechanism, often called recruitment, is an
  example of cooperative binding of proteins to DNA.

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Some activators work by allostery and regulate
steps after RNA polymerase binding
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• Not all promoters are limited in the same way .In
  some cases, closed complex does not spontaneously
  undergo transition to the open complex, so an
  activator must stimulate the transition from
  closed to open complex.
• Activators interact with the stable closed
  complex and induce a conformational change that
  causes transition to the open complex. This
  mechanism is an example of allostery.

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Action at a distance and DNA looping

• A. Cooperative binding of proteins to adjacent
  sites.
• B. cooperative binding of proteins to separated
  sites.

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DNA-bending protein can facilitate interaction
between DNA-bending proteins.
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Cooperative binding and allostery have many roles
in gene regulation

• Antitermination and beyond not all of gene
  regulation targets transcription initiation.

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Topic2 Regulation of transcription initiation
examples from bacteria
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• Now we have turn to some examples that show how
  these principles work in real cases.
• We will see how an activator and a repressor
  regulate expression in response to two signals,
  and also describe some of the experimental
  approaches that reveal how these regulators work.

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An activator and a repressor together control the
Lac genes

• These genes are expressed at high levels only
  when lactose is available, and glucose- the
  preferred energy source-is not.
• Two regulatory proteins are involved one is an
  activator called CAP, the other a repressor
  called the Lac repressor.
• Each of these respond environmental signal and
  communicates it to the Lac genes.

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The three Lac genes-LacZ, LacY, LacA-are arranged
adjacently on the E.coli genome and are called
the Lac operon. They are transcribed as a single
m RNA from the promoter.
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Expression of the Lac genes
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• The presence or absence of the sugars lactose and
  glucose control the level of the Lac genes. High
  levels of expression require the presence of
  lactose and absence of the preferred energy
  source, glucose. CAP and Lac repressor are shown
  as single units, but CAP actually binds DNA as a
  dimer, and Lac repressor binds as a tetramer. CAP
  recruits polymerase to the Lac promoter where it
  spontaneously undergoes isomerization to the open
  complex.

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Cap and Lac repressor have opposing effects on
RNA polymerase binding to the Lac promoter
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The site bound by Lac repressor is called the Lac
operator. The symmetric half-sites of the Lac
operator.
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• The Lac operator overlaps the promoter, and so
  repressor bound to the operator physically
  prevents RNA polymerase from binding to the
  promoter and thus initiating RNA synthesis.
• CAP binds as a dimer to a site similar in length
  to the Lac operator, but different in sequence.
  When CAP binds to that site, the activator helps
  polymerase bind to the promoter by interacting
  with the enzyme and recruiting it to the
  promoter.

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The control region of the Lac operon.The colored
bars above and below the DNA show regions covered
by RNA polymerase and the regulatory proteins.
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CAP has separate activating and DNA-binding
surfaces

• CAP activates the Lac genes by simple recruitment
  of RNA polymerase. Mutant versions of CAP have
  been isolated that bind DNA but do not activate
  transcription. (positive control)
• The amino acid substitutions in the positive
  control mutants identify the region of CAP that
  touches polymerase, called the activating region.

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RNA polymerase binding at the Lac promoter with
the help of CAP. CAP is recognized by the CTDs of
the aCTDs of the asubunits.Activation of the Lac
promoter by CAP
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Structure of CAP- aCTD-DNA complex.CAP is bound
as a dimer to its site. In this case, the aCTD of
RNA polymerase is bound to an adjacent stretch of
DNA, and interacting with CAP.
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CAP and Lac repressor bind DNA using a common
structure motif

• Although the details of DNA binding for bacterial
  activators and repressors differ (including CAP
  and the Lac repressor), the basic mechanism of
  DNA recognition is similar for most bacterial
  regulators.
• In the typical case, the protein binds as a
  homodimer to a site that is an inverted repeat.

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One monomer binds each half-site, with the axis
of symmetry of the dimer lying over that of the
binding site. Recognition of specific DNA
sequences is achieved using a conserved region of
secondary structure called a helix-turn helix.
This domain is composed of two ahelices, one of
which- the recognition helix.
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• Lac repressor binds as a tetramer to two
  operators. Each operator is contacted by only
  two of these subunits.

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Hydrogen bonds between ?repressor and base pairs
in the major groove of its operator.
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The active of Lac repressor and CAP are
controlled allosterically by their signals

• The conversion of lactose to allolactose is
  catalyzed by ß-galactosidase, itself encoded by
  one of the Lac genes.
• Allolacrose binds to Lac repressor and triggers a
  change in the shape of that protein.
• CAP activity is regulated in a similar manner.
  Only when glucose levels are low does CAP bind
  DNA and activate the Lac genes. Then c AMP
  is separate from the part of the protein that
  binds DNA.

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• Combinatorial control CAP controls other genes
  as well.
• Alternative sfactors direct RNA polymerase to
  alternative sets of promoters. (one of these
  alternatives is the heat shock sfactor, s32
  another example of an alternativesfactor, s54.

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NtrC and MerR transcriptional activators that
work by allostery rather than by recruitment.

• NtrC controls expression of genes involved in
  nitrogen metabolism, such as the glnA gene.
• MerR controls a gene called merT, which encodes
  an enzyme that makes cells resistant to the toxic
  effects of mercury. MerR also acts on an inactive
  RNA polymerase-promoter complex.

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Alternative sfactors control the ordered
expression of genes in a bacterial virus.The
bacterial phage SPO1 uses three sfactors in the
succession to regulate expression of its genome.
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NtrC has ATPase activity and works from DNA sites
far from the gene.

• Activation by NtrC. As with CAP, NtrC has
  separate activating and DNA-binding domains and
  binds DNA only in the presence of a specific
  signal.

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MerR activates transcription by twisting promoter
DNA.

• When bound to a single DNA-binding site, in the
  presence of mercury, MerR actives the merT gene.

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Some repressors hold RNA polymerase at the
promoter rather than excluding it

• Structure of a merT-like promoter.
• a. promoter with a 19bp spacer.
• b. promoter with a 19bp spacer when in complex
  with active activator.
• c. promoter with a 17bp spacer.

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AraC and control of the araBAD operon by
antiactivation

• Control of the araBAD operon.
• The promoter of the araBAD operon from E.coli is
  activated in the presence of arabinose and the
  absence of glycose and directs expression of
  genes encoding enzymes required for arabinose
  metabolism.two activators work together here
  AraC and CAP.

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• Topic3 examples of gene regulation at steps
  after transcription initiation

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Amino acid biosynthetic operons are controlled by
premature transcription termination

• In E.coli the five contiguous trp genes encode
  enzymes that synthesize the amino acid tryptophan.

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Transcription termination at the trp attenuator

• 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.

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Trp operator leader RNA
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• There is a second hairpin that can form between
  region1 and 2 of the leader
• Region 2 also is complementary to region 3 thus,
  yet another hairpin consisting of region2 and 3
  can form, and when it does it prevents the
  terminator hairpin from forming

• The leader RNA contains an open-reading frame
  encoding a short leader peptide of 147 amino
  acids, and this open-reading frame is preceded by
  a strong ribosome binding site

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Ribosomal proteins are translational repressors
of their own synthesis

• Regulation of translation often works in a manner
  analogous to transcriptional repression a
  repressor binds to the translation start site
  and blocks initiation of that process.

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• Control of ribosomal protein genes is simplified
  by their organization into several operons, each
  containing genes for up to 11 ribosomal proteins.
• E.coli ribosomal protein operons

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The primary control of ribosomal protein
synthesis is at the level of translation of the
mRNA.

• When extra copies of a ribosomal protein operon
  are introduced into the cell, the amount of mRNA
  increases. The cell compensates for extra mRNA by
  curtailing its activity as a template.
• For each operon, one( or a complex of two)
  ribosomal proteins binds the messenger near the
  translation initiation sequence of one of the
  first genes in the operon, preventing ribosomes
  from binding and initiating translation.

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Ribosomal protein S8 binds 16S rRNA

• How one protein can function both as a ribosomal
  component and as a regulator of its own
  translation is shown by comparing the sites where
  that protein binds to ribosomal RNA and to its
  messenger RNA.

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• The comparison suggests a precise mechanism of
  regulation. The binding site in the messenger
  includes the initiating AUG, mRNA bound by excess
  protein S8 cannot attach to initiate translation.
  Binding is stronger to ribosomal RNA than to
  mRNA, so translation is repressed only when all
  need for the protein in ribosome assembly is
  satisfied.

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Topic4 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.

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Alternative patterns of gene expression control
lytic and lysogenic growth

• ? has a 50-kb genome and some 50 genes. Most of
  these encode coat proteins, proteins involved in
  DNA replication, recombination and lysis.

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Promoters in the right and left control regions
of phage?

• The depicted region contains two genes( cI and
  cro) and three promoters. (PR, PL, PRM)

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Transcription in the? control regions in lytic
and lysogenic growth

• Two arrangements of gene expressin depicted.

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Regulatory proteins and their binding sites

• The cI encodes?repressor, a protein of two
  domains joined by a flexible linker region.

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Relative positions of promoter and operator sites
in OR
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?Repressor Binds to Operator Sites Cooperatively

• The ? repressor monomers interact to form dimers,
  and those dimers interact to form tetramers.These
  interaction ensure that binding of repressor to
  DNA is cooperative.

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Resspressor and Cro bind in Different Patterns to
Control Lytic and Lysogenic Growth

• How do repressor and Cro control the different
  patterns of gene expression associated with the
  different ways ? can grow?

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• For lytic growth , a single Cro dimer is bound to
  OR3 this site overlaps PRM and so Cro represses
  that promoter. PR binds RNA polymerase and
  directs transcription of lytic genes PL does
  likewise.
• During lysogeny, PRM is on, while PR and PL are
  off.repressor bound cooperatively at OR1and OR2
  blocks RNA polymerase binding at PR repressing
  transcription from that promoter.

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The action of ?repressor and Cro

• Repressor bound to OR1 and O R2 turns off
  transcription from PR.Repressor bound at OR2
  contacts RNA polymerase at PRM activating
  expression of the cIgene.OR3 lies within PRM Cro
  bound there represses transcription of cI.

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Lysogenic induction requires proteolytic cleavage
of ? repressor

• E.coli senses and responds to DNA damage. It does
  this by activating the function of a protein
  called RecA.
• Activated RecA stimulates autocleavage of LexA,
  releasing repression of those genes.this is
  called the SOS response.
• If the cell is a lysogen, ?repressor has evolved
  to resemble LexA, ensuring that ?repressor too
  undergoes autocleavage in response to activated
  RecA.

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The level of repressor in a lysogen must be
tightly regulated.

• Repressor ensures its level never drops toolow
  it activates its own expression ,an example of
  positive autoregulation.
• Repressor ensure its level never get too high,
  repressor also regulates itself negatively.(
  negative autoregulation )

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Negative Autoregulation of Repressor Requires
Long-Distance Interactions and a Large DNA
Loopinteraction of repressors at OR and OL
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Interactions between the C-terminal domain of
?repressors.
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Another Activator , ?c?, Controls the Decision
between Lytic and Lysogenic Growth upon Infection
of a New Host.Genes and prompters involved in
the lytic / lysogenic choice
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Growth Conditions of E.coli control the Stability
of C?protein and Lytic/Lysogenic
ChoiceEstablishment of lysogeny
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Transcriptional Antitermination in ?Development

• A example of gene regulation that operated at
  stages after transcription initiation, that we
  found in ?development, starting with a type of
  positive transcriptional regulation called
  antitermination.

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Recognition sites and sites of action of the ?N
and Q transcription antiterminator.
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Restroregulation An Interplay of Controls on Rna
Synthesis and Stability Determines int Gene
Expression DNA site and transcribed RNA
structures active in retroregulation of int
expression