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Bacteriophage lambda (l)

Bacteriophage lambda (l) . Transcriptional switches can regulate cellular decisions. Lysis or Lysogeny. Lysis : Infection by phage produces many progeny and breaks open (lyses) the host bacterium

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Bacteriophage lambda (l)

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  1. Bacteriophage lambda (l) Transcriptional switches can regulate cellular decisions

  2. Lysis or Lysogeny • Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium • Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively • No progeny • No lysis of the host • Can subsequently lyse (lysogeny) • Bacteriophage lambda can do either.

  3. UV Induction Lysogeny Lysis

  4. The phage genome integrated into the host bacterial genome is a prophage. Bacterium carrying the prophage is a lysogen. Lysogens are immune to further infection by similar phage because the phage functions are repressed in trans. Induction of the lysogen leads to excision of the prophage, replication of the phage DNA, and lysis of the host bacterium. Elements of lysogeny

  5. gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR Genes are clustered by function in the lambda genome Late control Virus head &tail Recombination Control region Replication Lysis cos origin promoter operator terminator Not to scale!

  6. gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR N Cro Immediate early transcription Transcription by E. coli RNA polymerase initiates at strong promoters PR , PR’, and PL , and terminates at t’s. 6S RNA

  7. N N N Antitermination by N protein leads to early gene expression gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL PRM PR tR1 PRE tR2 PR‘ t6S tR3 6S RNA N protein Cro CIII CII Q protein Recombination proteins Replication proteins

  8. Q gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR Cro Cro Lytic cascade: Cro turns off cI, Q protein action leads to late gene expression Lytic functions Replication proteins Viral head & tail proteins

  9. Q gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR Cro Cro Late stage of lytic cascade High concentrations of Cro turn off PR and PL . Abundant expression from PR’. Lytic functions Viral head & tail proteins

  10. + + CIII CII CII gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR CI Int Lysogeny: CII and CIII stimulate expression of cI to make repressor tint PRE = promoter for repression establishment Repressor

  11. gam int red N cI cro cII O P Q S R A…J att xis cIII Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR CI CI CI Lysogeny: Repressor turns off transcription PRM = promoter for repression maintenance Repressor Activated by Repressor binding to oR1 & oR2

  12. Regulatory mutants of lambda Clear plaque mutants Need wild type for lysogeny: Establishment Maintenance cI Yes Yes cII Yes No cIII Yes No Act in trans Virulent mutants (vir) Act in cis : are double mutants in oR &/or oL

  13. l operators overlap promoters oR : oR3 oR2 oR1 PR -35 -10 TTGACT GATAAT cro N TTAGAT 5’ ATAGAT 5’ -10 -35 PRM

  14. Repressor structure l repressor is a dimer; monomer has 236 amino acids. l repressor can bind cooperatively to operator sub-sites.

  15. Place a convenient reporter gene under control of the regulatory elements being studied Use a known regulatory region to control the trans-acting regulatory element Use hybrid genes to dissect regulatory schemes

  16. l-lachybrid genes PlacelcI gene under lac control. Use lacZ as a reporter. lac p, o l cI l pR , OR lacZ 321 Control amount of l repressor by [IPTG]. E. coli with lac repressor, no lacZ. See effect of l repressor by b-galactosidase activity

  17. l repressor will turn off expression from PR & PL lac p, o l cI l pR , OR lacZ b-galactosidase l repressor [IPTG] l repressor acts cooperatively.

  18. Repressor stimulates transcription from PRM lac p, o l cI l pRM , OR lacZ 123 b-galactosidase l repressor [IPTG] l repressor at oR1 and oR2 stimulates transcription from pRM.

  19. Binding of repressor blocks transcription from pR but activates pRM PR -35 -10 2 dimers of Repressor, bound cooperatively oR3 RNA Pol cro N oR2 oR1 -10 -35 = operator PRM = promoter -35 -10

  20. lysis or lysogeny (cI or Cro?) ? Both lysis and lysogeny: PR, PL, PR’ active : synthesize N, Cro antitermination by N : synthesize cIII, cII, Q Lysis: Low [Cro] : binds OR3, shuts off PRM (cI) High [Cro] : shuts off PR and PL antitermination by Q + activation of PR’ by Cro Bacteriophage l: Events leading to lysis

  21. lysis or lysogeny (cI or Cro?) ? Lysis and lysogeny : PR, PL, PR’ active : synthesize N, Cro antitermination by N : synthesize cIII, cII, Q Lysogeny: cII stimulate expression from PRE (cI repressor) and PINT (integrase) cIII stabilizes cII cI repressor shuts off PR, PL, PR’ (no lytic functions), stimulates PRM Bacteriophage l: Events leading to lysogeny

  22. Temperate and lytic phage have a different plaque morphology Lytic phage: clear plaques

  23. Induction and immunity of lysogens l

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