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.
UV Induction
Lysis
Lysogeny
Lysogeny: CII and CIII stimulate expression of cI to
make repressor
+
CII
att
int
gam
red
xis cIII N
tint Pint
Int
+
CIII
tL1
cI
cro
CII
cII O P Q
PL oL PRM PR tR1 PRE
oR
CI
Repressor
SR
A…J
tR2 PR‘ t6S
tR3
PRE = promoter for
repression
establishment
Lysogeny: Repressor turns off transcription
CI
att
int
gam
red
xis cIII N
Pint
tL1
CI
cI
cro
cII O P Q
PL oL PRM PR tR1 PRE
oR
CI
Repressor
SR
A…J
tR2 PR‘ t6S
tR3
PRM = promoter for
repression
maintenance
Activated by Repressor
binding to oR1 & oR2
l operators overlap promoters
oR :
oR3
oR2
oR1
PR
-35
TTGACT
-10
GATAAT
cro
N
TTAGAT 5’
-10
ATAGAT 5’
-35
PRM
Repressor structure
l repressor is a dimer; monomer has 236 amino acids.
C-terminal domain: protein-protein interaction;
dimerization and cooperativity
Connector
N-terminus: DNA binding; Helix-Turn-Helix motif
operator
l repressor can bind cooperatively
to operator sub-sites.
operator
oR2
operator
oR1
l-lac hybrid genes
Place l cI gene under lac control.
lac p, o
l cI
Use lacZ as a reporter.
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
Repressor stimulates 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.
Binding of repressor blocks transcription from pR but
activates pRM
PR
-35
-10
oR3
N
2 dimers of
Repressor,
bound
cooperatively
cro
RNA Pol
-10
oR2
oR1
-35
= operator
PRM
-35
-10 = promoter
Events at
initiation of
transcription
Abortive initiation assay
Let R = RNA polymerase, P = promoter (closed), and Po=
promoter (open)
R+P
KB
RP
kf
kr
[ApUp*U]
ATP + UTP*
RPo
ApUp*U
lag
time
Abortive transcripts
Measure kf and KB from lag time vs. 1/[R]
Lag time in abortive initiation assay is inversely proportional to [R].
Lag time =
1
KB kf
x 1
[R]
Lag time
Y-intercept = 1
kf
+ 1
kf
Slope =
1
[R]
1
KB kf
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 of
λ Repressor
Glu
Glu
Glu
Mutations in the δ subunit of RNA
polymerase that interfere w/λ repressormediated 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
The Awesome Power of Genetics