Bacteriophage lambda (l)Lysis or LysogenyInfection by temperate phage leads to lysis or lysogenyTemperate and lytic phage have a different plaque morphologyElements of lysogenyInduction and immunity of lysogensRegulatory mutants of lambdaGenes are clustered by function in the lambda genomeImmediate early transcriptionAntitermination by N protein leads to early gene expressionLytic cascade: Cro turns off cI, Q protein action leads to late gene expressionLate stage of lytic cascadeLysogeny: CII and CIII stimulate expression of cI to make repressorLysogeny: Repressor turns off transcriptionl operators overlap promotersRepressor structureLambda repressor bound to DNA via a helix-turn-helix domainCro structureCompetition between repressor and Cro for operator sitesUse hybrid genes to dissect regulatory schemesl/lac hybrid genesl repressor will turn off expression from PR & PLMutation of oR1 decreases affinity for l repressorRepressor will stimulate transcription from PRMBinding of repressor blocks transcription from pR but activates pRMTwo repressor dimers interacting cooperatively via the C terminal domainBacteriophage l: Events leading to lysisBacteriophage l: Events leading to lysogenyFactors favoring lysogeny cause increased concentrations of repressor vs. CroHomework problems provide a quantitative approach to the competition between Cro and repressor for the l operators.Bacteriophage lambda () Transcriptional switches can regulate cellular decisionsLysis 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.Infection by temperate phage leads to lysis or lysogenyE. coli chromosomeλλ phageE. coli cellE. coli cell++lytic growth of phagecell undergoes lysogenyλ prophageTemperate and lytic phage have a different plaque morphologyTemperate phage generate turbid plaqueslysogenized cellslysed cellsuninfected cellslysed cellsMutants of phage that have lost the capacity to lysogenize form clear plaquesLytic phage: clear plaquesElements 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.Induction and immunity of lysogensλA λ lysogenλSpontaneously,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+Regulatory mutants of lambdaClear plaque mutantsVirulent mutants (vir)Need wild type for lysogeny:Establishment MaintenancecI Yes YescII Yes NocIII Yes NoAct in trans Act in cis : are double mutants in oR &/or oLGenes are clustered by function in the lambda genomeRecombination Control region Replication LysisVirus head&tailoriginoR Pint oL PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JpromoteroperatorterminatorLate controlcosNot to scale!Immediate early transcriptionTranscription by E. coli RNA polymerase initiates at strongpromoters PR , PR’, and PL , and terminates at t’s.6S RNAoR Pint oL PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JNCroAntitermination by N protein leads to early gene expressionPint PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JN N NN protein Cro6S RNACIIIRecombination proteinsCIIReplication proteinsQ proteinLytic cascade: Cro turns off cI, Q protein action leads to late gene expressionoR Pint oL PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JCro CroQLytic functionsReplication proteinsViral head & tail proteinsLate stage of lytic cascadeHigh 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 attintxisredgamcIIIN cI cro cII O P Q S R A…JCro Cro QLytic functionsViral head & tail proteins+Lysogeny: CII and CIII stimulate expression of cI to make repressoroR Pint oL PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JCIIICIICI+RepressorPRE = promoter for repressionestablishmentInttint CIILysogeny: Repressor turns off transcriptionoR Pint oL PL PRM PR PRE PR‘tR3 tL1 tR1 tR2 t6S attintxisredgamcIIIN cI cro cII O P Q S R A…JCIRepressorPRM = promoter for repressionmaintenanceCICIActivated by Repressorbinding to oR1 & oR2operators overlap promotersoR1oR2oR :TTGACT GATAAT-10-35ATAGAT 5’TTAGAT 5’-10 -35oR3cro N PR PRMRepressor structureoperatorN-terminus: DNA binding; Helix-Turn-Helix motifC-terminal domain: protein-protein interaction; dimerization and cooperativityConnectoroperatoroperatoroR1oR2 repressor is a dimer; monomer has 236 amino acids.repressor can bind cooperativelyto operator sub-sites.http://www.rtc.riken.go.jp/jouhou/image/dna-protein/all/small_N1lli.gifLambda repressor bound to DNA via a helix-turn-helix domainCro structureoperatorDNA binding; Helix-Turn-Helix motif; also dimerizationoR3Crois a dimerMonomer has 66 amino acids Has only one protein domainDoes NOT display cooperativityCompetition between repressor and Cro for operator sitesPRMPLPR-10-35-35-10-10 -35OR1OR2OR3OL1 OL2 OL3OL3OL1 OL2 OR3OR2OR1cINcroAffinity forRepressorCroHigh HighHigh HighHighHighLowLow LowLowLowLowUse 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/lachybrid geneslac p, o cI pR , OR 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 activity321 repressor will turn off expression from PR & PLlac p, o cI pR , OR lacZ -galactosidase[IPTG] repressor repressor acts cooperatively.Mutation of oR1 decreases affinity for  repressorlac p, o cI pR , OR lacZ