Bacteriophage_lambda_.ppt
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The document describes the life cycle choices - lysis vs lysogeny - of bacteriophage lambda that infects E. coli. During lysis, the phage DNA replicates and causes the host cell to burst, releasing progeny virus. In lysogeny, the phage DNA integrates into the host genome and remains dormant. Gene expression is regulated by transcriptional switches that determine whether lytic or lysogenic development occurs.
![lac p, o l cI l pR , OR lacZ
Place l cI gene under lac control. Use lacZ as a reporter.
E. coli with lac repressor,
no lacZ.
Control amount of l
repressor by [IPTG].
See effect of l repressor
by b-galactosidase activity
321](https://image.slidesharecdn.com/14349bacteriophagelambdalecture11-221005082608-d763375e/85/Bacteriophage_lambda_-ppt-16-320.jpg)
![lac p, o l cI l pR , OR lacZ
b-galactosidase
[IPTG]
l repressor
l repressor acts cooperatively.](https://image.slidesharecdn.com/14349bacteriophagelambdalecture11-221005082608-d763375e/85/Bacteriophage_lambda_-ppt-17-320.jpg)
![lac p, o l cI l pRM , OR lacZ
b-galactosidase
[IPTG]
l repressor
123
l repressor at oR1 and oR2 stimulates transcription from pRM.](https://image.slidesharecdn.com/14349bacteriophagelambdalecture11-221005082608-d763375e/85/Bacteriophage_lambda_-ppt-18-320.jpg)
![ 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](https://image.slidesharecdn.com/14349bacteriophagelambdalecture11-221005082608-d763375e/85/Bacteriophage_lambda_-ppt-20-320.jpg)
Bacteriophage_lambda_.ppt
- 1. 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.
- 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.
- 5. Genes are clustered by function in the lambda genome Recombination Control region Replication Lysis Virus head &tail origin oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J promoter operator terminator Late control cos Not to scale!
- 6. Immediate early transcription Transcription by E. coli RNA polymerase initiates at strong promoters PR , PR’, and PL , and terminates at t’s. 6S RNA oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J N Cro
- 7. Antitermination by N protein leads to early gene expression Pint PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J N N N N protein Cro 6S RNA CIII Recombination proteins CII Replication proteins Q protein
- 8. Lytic cascade: Cro turns off cI, Q protein action leads to late gene expression oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J Cro Cro Q Lytic functions Replication proteins Viral head & tail proteins
- 9. Late stage of lytic cascade High concentrations of Cro turn off PR and PL . Abundant expression from PR’. oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J Cro Cro Q Lytic functions Viral head & tail proteins
- 10. + Lysogeny: CII and CIII stimulate expression of cI to make repressor oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J CIII CII CI + Repressor PRE = promoter for repression establishment Int tint CII
- 11. Lysogeny: Repressor turns off transcription oR Pint oL PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S att int xis red gam cIII N cI cro cII O P Q S R A…J CI Repressor PRM = promoter for repression maintenance CI CI Activated by Repressor binding to oR1 & oR2
- 12. Regulatory mutants of lambda Clear plaque mutants Virulent mutants (vir) Need wild type for lysogeny: Establishment Maintenance cI Yes Yes cII Yes No cIII Yes No Act in trans Act in cis : are double mutants in oR &/or oL
- 13. oR1 oR2 oR : TTGACT GATAAT -10 -35 ATAGAT 5’ TTAGAT 5’ -10 -35 oR3 cro N PR PRM
- 14. operator N-terminus: DNA binding; Helix-Turn-Helix motif C-terminal domain: protein-protein interaction; dimerization and cooperativity Connector operator operator oR1 oR2 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
- 16. lac p, o l cI l pR , OR lacZ Place l cI gene under lac control. Use lacZ as a reporter. E. coli with lac repressor, no lacZ. Control amount of l repressor by [IPTG]. See effect of l repressor by b-galactosidase activity 321
- 17. lac p, o l cI l pR , OR lacZ b-galactosidase [IPTG] l repressor l repressor acts cooperatively.
- 18. lac p, o l cI l pRM , OR lacZ b-galactosidase [IPTG] l repressor 123 l repressor at oR1 and oR2 stimulates transcription from pRM.
- 19. oR1 oR2 -10 -35 -10 -35 oR3 cro N PR PRM 2 dimers of Repressor, bound cooperatively RNA Pol = operator -10 -35 = promoter
- 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
- 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
- 22. Temperate and lytic phage have a different plaque morphology Temperate phage generate turbid plaques lysogenized cells lysed cells uninfected cells lysed cells Mutants of phage that have lost the capacity to lysogenize form clear plaques Lytic phage: clear plaques
- 23. Induction and immunity of lysogens l A l lysogen l Spontaneously, 1/1000 lysogens will induce, i.e. the l prophage will excise, replicate and lyse the cell. UV treatment leads to induction of virtually all lysogens in a culture. Lysogens are immune to further infection with similar (lambdoid) phage + l