Transcript 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.
UV Induction
Lysis
Lysogeny
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.
Genes are clustered by function in the
lambda genome
Late control
Recombination
att
int
Control region Replication
gam
red
xis cIII N
Pint
tL1
cI
cro
cII O P Q
Virus head
Lysis &tail
SR
A…J
PL oL PRM PR tR1 PRE tR2 PR‘ t6S cos
tR3
oR
origin
promoter
operator
terminator
Not to scale!
Immediate early transcription
Transcription by E. coli RNA polymerase initiates at strong
promoters PR , PR’, and PL , and terminates at t’s.
att
int
gam
red
xis cIII N
Pint
tL1
cI
cro
cII O P Q
PL oL PRM PR tR1 PRE
oR
SR
tR2 PR‘ t6S
tR3
6S RNA
N
Cro
A…J
Antitermination by N protein leads to early
gene expression
N
att
int
gam
red
xis cIII N
Pint
tL1
N
cI
PL
cro
N
cII O P Q
PRM PR tR1 PRE
SR
A…J
tR2 PR‘ t6S
tR3
6S RNA
N protein
CIII
Recombination proteins
Cro
CII
Q protein
Replication proteins
Lytic cascade: Cro turns off cI, Q protein
action leads to late gene expression
Cro
att
int
gam
red
xis cIII N
Pint
tL1
Cro
cI
cro
Q
cII O P Q
PL oL PRM PR tR1 PRE
oR
SR
A…J
tR2 PR‘ t6S
tR3
Lytic functions
Replication proteins
Viral head & tail proteins
Late stage of lytic cascade
High concentrations of Cro turn off PR and PL .
Abundant expression from PR’.
Cro
att
int
gam
red
xis cIII N
Pint
tL1
Cro
cI
Q
cro
cII O P Q
PL oL PRM PR tR1 PRE
oR
SR
A…J
tR2 PR‘ t6S
tR3
Lytic functions
Viral head & tail proteins
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
Regulatory mutants of lambda
Clear plaque mutants
Need wild type for lysogeny:
cI
Establishment
Yes
Maintenance
Yes
cII
Yes
No
cIII
Yes
No
Act in trans
Virulent mutants (vir)
Act in cis : are double mutants in oR &/or oL
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-te r m inal dom ain: prote in-prote in inte r action;
dim e rization and coope r ativity
Conne ctor
N-te r m inus : DNA binding; He lix-Tur n-He lix m otif
ope rator
l repressor can bind cooperatively
to operator sub-sites.
ope rator
oR2
ope rator
oR1
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
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
l repressor will turn off expression from PR & PL
lac p, o
l cI
l pR , OR
b-galactosidase
[IPTG]
l repressor acts cooperatively.
lacZ
l repressor
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
Bacteriophage l: 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
Bacteriophage l: 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
Temperate and lytic phage have a different
plaque morphology
Mutants of phage that
have lost the capacity to
lysogenize form clear
plaques
Temperate phage
generate turbid
plaques
lys oge nize d ce lls
lys e d ce lls
lys e d ce lls
uninfe cte d ce lls
Lytic phage: clear plaques
Induction and immunity of lysogens
l
A l lysogen
Spontaneously,
1/1000 lysogens w ill
induce ,i.e. the ll
prophage w ill
excise, replicate and
lyse the cell.
+
UV treatment leads
to inductionof
virtually all lysogens
in a culture.
Lysogens areim m uneto
f urther infection w ith similar
(lambdoid) phage
l