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Molecular Biology 1 BC 21C

Molecular Biology 1 BC 21C . Biology and Genetics of Bacteriophage Lambda. Lambda genetic map.

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Molecular Biology 1 BC 21C

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  1. Molecular Biology 1BC 21C Biology and Genetics of Bacteriophage Lambda

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Delayed Early • Transcription occurs from PR to either tR2 or tR3 and from PL to tL2. • PL to tL2 transcribes genes:1) 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

  8. Delayed Early • PR to tR3 transcribes genes:1) 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.

  9. 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 >> OR2 = OR1Cro affinity: OR3 > OR3+OR2 or OR3+OR1 > OR3+OR2+OR1

  10. 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.

  11. 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.

  12. 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.

  13. 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).

  14. 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 >>> OR3

  15. 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----

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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 mutants:lind-: pcI mutants that can't be cleaved during SOSlcI-: no pcI madelvir : mutant OR and OL so pcI can't bind

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