Molecular Biology 1 BC 21C

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Molecular Biology 1BC 21C

• Biology and Genetics of Bacteriophage Lambda

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Lambda genetic map

• Temperate phage. Genome linear in phage, circular
  in host. Circularizes by cos site overhangs of 12
  basesLytic three phases - immediate early,
  delayed early and late lysis. Replication of the
  lambda genome, production of viral coat proteins,
  and assembly of progeny phage.

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Lambda genetic map

• Lysogenic three phases
• immediate early,
• delayed early and
• late lysogeny.
• Repressor protein pcI is produced which binds to
  the two operator regions, OR and OL.
• Transcription of all lambda genes except cI is
  stopped.
• No progeny produced. Phage genome integrates into
  host genome and becomes prophage. Not
  disadvantageous for host.

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Lambda genetic map

• The health of the host cell determines the fate
  of lambda.
• If the cell is healthy, sufficient energy is
  available for production of progeny phage and
  lambda will go lytic.
• If the cell is unhealthy, lambda will go
  lysogenic.

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Immediate Early

• Host RNAP transcribes from PR to tR1 and PL to
  tL1.
• The lambda genes cro and N are transcribed.cro
  product is Cro.
• Cro is a repressor that binds to DNA at the
  operator sites. It is negatively autoregulated.
• Cro is called the antirepressor because it
  competes with pcI, the lambda repressor.

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Immediate Early

• N product is pN. pN is an antiterminator that
  prevents transcription termination at tR1 and
  tL1. pN binds to the RNA transcripts from PR and
  PL at the nutR and nutL (N-utilization) sites,
  respectively.
• This prevents the stem-loop of tR1 and tL1 from
  forming thus, tR1 and tL1 are no longer
  recognized by RNAP as terminators and
  transcription continues from PR to either tR2 or
  tR3 and from PL to tL2.

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Delayed Early

• Transcription occurs from PR to either tR2 or
  tR3 and from PL to tL2.
• PL to tL2 transcribes genes1) N Produces pN,
  the antiterminator
• 2) cIII Produces pcIII, a chaperone protein
  that protects pcII from host proteases
• 3) xis Produces excisionase, an endonuclease
  required for excision of the lambda prophage
• 4) int Produces integrase, an endonuclease
  required for both integration and excision of
  lambda
• 5) attP site The site of integration via
  homologous recombination with the bacterial attB
  site
• 6) sib site This site forms a stem-loop
  structure near the 3' end of the transcript that
  is a target for host RNase

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Delayed Early

• PR to tR3 transcribes genes1) cro Produces
  Cro, a repressor called the antirepressor.
• 2) cII Produces pcII, an activator protein that
  binds to the lambda DNA and activates three
  promoters (PI, PRE, and Panti-Q)
• 3) O and P Required for theta replication of
  lambda DNA. In late lysis, production of these
  proteins stops and rolling circle replication of
  lambda begins.
• 4) Q Produces pQ, another antiterminator that
  binds to RNA at the qut site located near tR4.
  Antitermination of tR4 allows transcription from
  PR2 (also called PR') to proceed through to tR5,
  thus transcribing all of the late lamba
  structural genes required for phage assembly.
• Both Cro and pcI can bind to the operator sites
  OR and OL. The operator sites are actually
  composed of three specific operator regions OR1,
  OR2 and OR3.

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Late Lysis

• Occurs in a healthy host.
• 1) pcII is degraded by host proteases HflA/HflB
  that are only made in a healthy host. PRE
  (promoter for repressor establishement) is not
  activated so the lambda repressor pcI is not
  produced.
• 2) Cro binds to the operator sites to repress
  transcription from PRM (promoter for repressor
  maintenance) that is necessary for continued
  transcription of cI.
• Cro has the highest affinity for OR3/OL3 and will
  bind to these regions first to repress PRM.
• As Cro accumulates, it also binds to OR2/OL2 then
  OR1/OL1 to repress PR and PL. Cro binding is not
  cooperative OR3 gtgt OR2 OR1Cro affinity OR3 gt
  OR3OR2 or OR3OR1 gt OR3OR2OR1

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Late Lysis

• 3) RNA transcript from PL to tL2 has the sib
  region at the 3' end. The sib region forms a
  stem-loop structure in the RNA molecule that is
  targeted by host RNases and the RNA is degraded
  3' to 5' which degrades the int ORF and reduces
  the amount of integrase produced.
• 4) Lambda DNA replicates via rolling circle
  replication to produce many copies of the lambda
  genome for packaging.
• 5) Transcription from PR2 to tR5 due to
  antitermination of tR4 by pQ. The late genes
  encoding the structural proteins (head and tail)
  and lysis proteins are transcribed and
  translated. Lambda progeny are assembled and
  released by lysis of the host cell.

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Late Lysogeny

• Occurs in an unhealthy host
• 1) Host proteases HflA/HflB are not produced in
  an unhealthy cell and so pcII is not degraded.
  pcII activates transcription from three
  promoters PRE, PI and Panti-Q.
• 2) Transcript from PI contains only the int ORF
  for increased production of integrase, the
  recombinase required for integration of the
  lambda genome into the E. coli chromosome.

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Late Lysogeny

• 3) Transcription from Panti-Q produces the Q
  antisense RNA, an RNA molecule that is able to
  bind to the Q ORF present on the PR to tR3
  transcript and prevent translation of Q.
• This halts production of the pQ antiterminator
  and transcription from PR2 proceeds only to the
  tR4 terminator. Thus, transcription of the
  structural protein genes is prevented.
• 4) Transcript from PRE contains the cro antisense
  and the pI ORF. The 5' end of this transcript,
  containing the cro antisense RNA, is able to bind
  to the cro ORF present on the PR transcripts and
  prevent translation of Cro, the antirepressor.
• Thus, Cro protein concentrations drop and Cro is
  unable to compete with pcI for binding of the
  operator sites.

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Late Lysogeny

• 5) pcI protein is produced from the PRE
  transcript and is able to bind to the operator
  sites. pcI is made up of two subunits which form
  a "dumbbell" with one end the DNA binding end and
  the other end the repressor binding end. pcI
  forms a dimer to bind DNA. pcI has the highest
  affinity for OR1/OL1, but binding to OR1 and OR2
  is cooperative. So at low pcI concentrations it
  binds to both OR1/OL1 and OR2/OL2. Binding of pcI
  to OR1 represses PR (and PL by binding OL1).

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5) continued

• Binding of pcI to OR2 activates transcription of
  PRM, the promoter for repressor maintenance, that
  transcribes only the cI gene. Thus, pcI is a
  positive autoregulator. At high pcI
  concentrations, pcI also binds to OR3/OL3.
  Binding of pcI to OR3 represses transcription of
  PRM, so pcI is also a negative autoregulator.pcI
  affinity OR1 OR2 gtgtgt OR3

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• 6) The lambda DNA integrates into the host
  chromosome by homologous recombination at the
  attP site (POP') of lambda and the attB (BOB')
  site of E. coli to form the prophage. In the
  lysogen, the only lambda protein produced is pcI
  by transcription from PRM and translation of the
  cI ORF.
• ----gal----BOP'--------l-------------POB'-----bio-
  ---

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Induction

• Induction involves excision of the lambda genome
  from the host chromosome followed by the lytic
  cycle. The premise of induction is that lambda
  will escape from a damaged cell rather than die
  along with it. In other words, lambda is like a
  rat jumping off a sinking ship.

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Induction

• Lambda is able to sense that the host cell is
  struggling when the host enters into SOS. RecA
  protease activity will catalyze the autolysis of
  pcI. The cleaved pcI protein is unable to bind to
  the operator sites and so PR and PL are
  derepressed and PRM is no longer activated.
  Transcription from PR and PL signals the start of
  the immediate early infection cycle and so begins
  the lytic life cycle. Lambda progeny are
  assembled and released from the host cell.

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Induction

• Why must the pcI repressor negatively
  autoregulate its own expression? Because if the
  concentration of pcI is too high, not all of the
  pcI will be cleaved by RecA and induction will
  not occur.During induction, both excisionase and
  integrase are required for excision of lambda
  from the host chromosome.
• Both are required for excision because integrase
  recognizes BOP' but not POB' so excisionase is
  needed for both sites to be recognized and
  recombined.

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Induction

• Since the attP site is located between int and
  sib, the mRNA produced from PL past tL1 includes
  xis and int but not sib. Thus, there is no
  degradation of the PL transcript and both
  integrase and excisionase are produced.

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Immunity to superinfection

• Any lambda DNA entering a lysogen will
  immediately be bound by the pcI made by the
  existing prophage. Thus, PR and PL of the
  incoming lambda are immediately repressed by pcI
  and the new lambda DNA is never expressed.

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Zygotic induction

• When an Hfr donor transfers a lambda prophage to
  a non-lysogenic recipient, there is immediate
  induction because there is no pcI in the
  recipient.
• Some lambda mutantslind- pcI mutants that
  can't be cleaved during SOSlcI- no pcI
  madelvir mutant OR and OL so pcI can't bind