1. Bacteriophage lambda ( ) 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

7. Regulatory mutants of lambda Clear plaque mutants Virulent mutants (vir) Need wild type for lysogeny: EstablishmentMaintenance cIYes cIIYesNo cIIIYesNo Act in trans Act in cis : are double mutants in o R &/or o L

8. Genes are clustered by function in the lambda genome RecombinationControl regionReplicationLysis Virus head &tail origin oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J promoter operator terminator Late control cos Not to scale!

9. Immediate early transcription Transcription by E. coli RNA polymerase initiates at strong promoters P R, P R ’, and P L, and terminates at t’s. 6S RNA oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J N Cro

10. Antitermination by N protein leads to early gene expression P int PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J NNN N proteinCro 6S RNA CIII Recombination proteins CII Replication proteins Q protein

11. Lytic cascade: Cro turns off cI, Q protein action leads to late gene expression oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J Cro Q Lytic functions Replication proteins Viral head & tail proteins

12. Late stage of lytic cascade High concentrations of Cro turn off P R and P L. Abundant expression from P R ’. oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J Cro Q Lytic functions Viral head & tail proteins

13. + Lysogeny: CII and CIII stimulate expression of cI to make repressor oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J CIII CII CI + Repressor P RE = promoter for repression establishment Int t int CII

14. Lysogeny: Repressor turns off transcription oRoR P int oLoL PLPL P RM PRPR P RE PR‘PR‘ t R3 t L1 t R1 t R2 t 6S att int xis red gam cIII NcIcrocIIOPQSRA…J CI Repressor P RM = promoter for repression maintenance CI Activated by Repressor binding to o R1 & o R2

15.  operators overlap promoters oR1oR1oR2oR2 o R : TTGACTGATAAT -10-35 ATAGAT 5’TTAGAT 5’ -10-35 oR3oR3 cro N PRPR P RM

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

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

18. Cro structure Cro  is 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. /lac  hybrid genes lac p, o  cI  p R, O R lacZ Place cI gene under lac control. Use lacZ as a reporter. E. coli with lac repressor, no lacZ. Control amount of  repressor by [IPTG]. See effect of  repressor by  -galactosidase activity 321

22. repressor will turn off expression from P R & P L lac p, o  cI  p R, O R lacZ  -galactosidase [IPTG] repressor repressor acts cooperatively.

23. Mutation of o R 1 decreases affinity for repressor lac p, o  cI  p R, O R lacZ  -galactosidase [IPTG] repressor LOF mutation at o R 1

24. Repressor will stimulate transcription from P RM lac p, o  cI  p RM, O R lacZ  -galactosidase [IPTG] repressor 123 repressor at o R 1 and o R 2 stimulates transcription from p RM.

25. Binding of repressor blocks transcription from p R but activates p RM oR1oR1oR2oR2 -10-35 -10-35 oR3oR3 cro N PRPR P RM 2 dimers of Repressor, bound cooperatively RNA Pol = operator -10-35 = promoter

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

27. Bacteriophage : Events leading to lysis lysis or lysogeny (cI or Cro?) ? Both lysis and lysogeny: –P R, P L, P R’ active : synthesize N, Cro –antitermination by N : synthesize cIII, cII, Q Lysis: –Low [Cro] : binds O R 3, shuts off P RM (cI) –High [Cro] : shuts off P R and P L –antitermination by Q + activation of P R’ by Cro

28. Bacteriophage : Events leading to lysogeny lysis or lysogeny (cI or Cro?) ? Lysis and lysogeny : –P R, P L, P R’ active : synthesize N, Cro –antitermination by N : synthesize cIII, cII, Q Lysogeny: –cII stimulate expression from P RE (cI repressor) and P INT (integrase) –cIII stabilizes cII –cI repressor shuts off P R, P L, P R’ (no lytic functions), stimulates P RM

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 P RE. –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 operators.