Download - Bacteriophage Lambda Lecture 1
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Bacteriophage lambda (l)
Transcriptional switches can regulate cellular decisions
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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.
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Lysis Lysogeny
UV Induction
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• 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
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Genes are clustered by function in the lambda genome
Recombination Control region Replication LysisVirus head&tail
originoR Pint oL PL PRM PR PRE PR‘
tR3 tL1 tR1 tR2 t6S
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
promoteroperatorterminator
Late control
cos
Not to scale!
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Immediate early transcriptionTranscription by E. coli RNA polymerase initiates at strongpromoters PR , PR’, and PL , and terminates at t’s.
6S RNA
oR Pint oL PL PRM PR PRE PR‘
tR3 tL1 tR1 tR2 t6S
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
N Cro
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Antitermination by N protein leads to early gene expression
Pint PL PRM PR PRE PR‘tR3
tL1 tR1 tR2 t6S
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
N N N
N protein Cro6S RNA
CIII
Recombination proteins
CII
Replication proteins
Q protein
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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
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
Cro Cro Q
Lytic functionsReplication proteinsViral head & tail proteins
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Late 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
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
Cro Cro Q
Lytic functionsViral head & tail proteins
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Lysogeny: CII and CIII stimulate expression of cI to make repressor
oR Pint oL PL PRM PR PRE PR‘
tR3 tL1 tR1 tR2 t6S
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
CIII CII
CI
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Repressor
PRE = promoter for repressionestablishmentInt
tint
CII
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Lysogeny: Repressor turns off transcription
oR Pint oL PL PRM PR PRE PR‘
tR3 tL1 tR1 tR2 t6S
attint
xisred
gam
cIII N cI cro cII O P Q S R A…J
CI
Repressor
PRM = promoter for repressionmaintenance
CI
CIActivated by Repressorbinding to oR1 & oR2
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Regulatory mutants of lambdaClear 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
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operators overlap promotersoR1oR2oR :
TTGACT GATAAT-10-35
ATAGAT 5’TTAGAT 5’-10 -35
oR3
cro N
PR
PRM
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Repressor structure
operator
N-terminus: DNA binding; Helix-Turn-Helix motif
C-terminal domain: protein-protein interaction; dimerization and cooperativity
Connector
operator operatoroR1oR2
repressor is a dimer; monomer has 236 amino acids.
repressor can bind cooperativelyto operator sub-sites.
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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
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-lachybrid genes
lac 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 activity
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repressor will turn off expression from PR & PL
lac p, o cI pR , OR lacZ
-galactosidase
[IPTG]
repressor
repressor acts cooperatively.
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Repressor stimulates transcription from PRM
lac p, o cI pRM , OR lacZ
-galactosidase
[IPTG]
repressor
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repressor at oR1 and oR2 stimulates transcription from pRM.
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Binding of repressor blocks transcription from pR but activates pRM
oR1oR2
-10-35
-10 -35
oR3
cro N
PR
PRM
2 dimers of Repressor, bound cooperatively
RNA Pol
= operator
-10-35 = promoter
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Bacteriophage : 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
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Bacteriophage : 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
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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
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Induction and immunity of lysogens
A lysogen
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
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