Bacteriophage lambda ( l )

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 • Bacteriophage lambda can do either.

3. Infection by temperate phage leads to lysis or lysogeny

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

5. Elements of lysogeny • 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.

6. Induction and immunity of lysogens l

7. 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

8. 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!

9. 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

10. 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

11. 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

12. 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

13. + + 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

14. 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

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

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

17. Lambda repressor bound to DNA via a helix-turn-helix domain http://www.rtc.riken.go.jp/jouhou/image/dna-protein/all/small_N1lli.gif

18. Cro structure Crois a dimer Monomer has 66 amino acids Has only one protein domain Does NOT display cooperativity

19. Competition between repressor and Cro for operator sites

20. Use hybrid genes to dissect regulatory schemes • 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

21. 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

22. 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.

23. Mutation of oR1 decreases affinity for l repressor lac p, o l cI l pR , OR lacZ LOF mutation at oR1 b-galactosidase l repressor [IPTG]

24. Repressor will stimulate 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.

25. 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

26. Two repressor dimers interacting cooperatively via the C terminal domain

27. Bacteriophage l: Events leading to lysis • 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

28. Bacteriophage l: Events leading to lysogeny • 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

29. Factors favoring lysogeny cause increased concentrations of repressor vs. Cro • High multiplicity of infection • More templates produce more of the CII protein, which stimulates PRE. • Phage sense that it is too crowded. • Poor nutrient conditions for host • Low [glucose] leads to increase in [cAMP]. • Increased [cAMP] will repress the host gene hflA. • Less HflA (a protease) leads to less degradation of the CII protein.

30. Homework problems provide a quantitative approach to the competition between Cro and repressor for the l operators.