Bacteriophage lambda (l)
description
Transcript of Bacteriophage lambda (l)
Bacteriophage lambda (l)
Transcriptional switches can regulate cellular decisions
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.
Lysis Lysogeny
UV Induction
+
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
+
Repressor
PRE = promoter for repressionestablishmentInt
tint
CII
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
operators overlap promoters
oR1oR2oR :
TTGACT GATAAT-10-35
ATAGAT 5’TTAGAT 5’-10 -35
oR3
cro N
PR
PRM
Repressor structure
operator
N-terminus: DNA binding; Helix-Turn-Helix motif
C-terminal domain: protein-protein interaction; dimerization and cooperativity
Connector
operator operator
oR1oR2
repressor is a dimer; monomer has 236 amino acids.
repressor can bind cooperativelyto operator sub-sites.
-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
321
Repressor stimulates transcription from PRM
lac p, o cI pRM , OR lacZ
-galactosidase
[IPTG]
repressor
123
repressor at oR1 and oR2 stimulates transcription from pRM.
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
Events at initiation of
transcription
Abortive initiation assay
Let R = RNA polymerase, P = promoter (closed), and Po= promoter (open)
R + P RP RPoATP + UTP*
ApUp*U
kf
kr
KB
Abortive transcripts
[ApUp*U]
time
lag
Measure kf and KB from lag time vs. 1/[R]
Lag time
Lag time in abortive initiation assay is inversely proportional to [R].
Lag time = KB kf
1
kf
1x +1
[R]
1
[R]Y-intercept = kf
1
Slope = KB kf
1
Effect of wild-type and pc mutant λ repressors on activity of PR & PRM
Effect of Operator Mutations on Transcriptional Control of PR&PRM
OR1-OR2+OR3- OR1+OR2-OR3+
Effect of λ-pc mutations on KB and k2
Architecture of λOR
Mutations in the Activating Region ofλ Repressor
Glu
Glu
Glu
Mutations in the δ subunit of RNA polymerase that interfere w/λ repressor-mediated activation of PRM transcription
Effect of mutations in the δ subunit of RNA polymerase on activator-dependent and independent transcription of the lac
promoter
A model for interaction of the δ subunit of RNA polymerase with λ repressor
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