Determination of l Phage Cycles Upon Infection of E. coli

1
Determination of l Phage Cycles Upon Infection of
E. coli

• Mike Gleason
• University of Nebraska Medical Center

2
Initial Infection
Cro
cII
O
P
int
xis
cIII
N
cI
Upon infection, here is what the injected l phage
genome looks like. All genes are turned off
until host transcription machinery binds to the
phage DNA.
3
Immediate Early Genes
PR
STOP
cII
O
P
Cro
int
xis
cIII
cI
N
STOP
PL
Products N, Cro
The hosts RNA polymerase has now started
transcribing from both the PL and PR promoters.
This results in production of the antiterminator
(N) protein and some Cro protein. At this time,
both transcripts terminate early due to the
presence of a rho-independent terminator sequence.
4
Early Genes
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
PL
Products N, cIII, xis, int, Cro, cII, O, P
The antiterminator (N) protein allows both PL and
PR to be read through, producing longer
transcripts. Since prokaryotes have
polycistronic mRNAs, each longer transcript can
now code for multiple proteins.
5
Decision Time
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
PL
Products N, cIII, xis, int, Cro, cII, O, P
The virus now uses the hosts relative health to
decide whether to enter the lytic phase or
lysogeny. If there is ample glucose available to
the host, cAMP is exported and cAMP is low.
Supplies of cAMP determines whether it can bind
and inactivate the host proteolytic enzyme HflA.
6
Lysis
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
Cro
Cro
PR
PRM
OR3
OR2
OR1
PRM
PL
Cro
Cro
Products Cro, cII, O, P
Glucose was in ample supply, so cAMP is exported
and cAMP is low. cAMP does not bind HflA, so
HflA is free to degrade cII, and therefore not
lead to cI. The site between the PR and PRM is
the operator OR. It has three similar cis
elements, OR1, OR2, and OR3, each of which can be
bound by Cro or cI. There are slight differences
in the sequences, so Cro has highest affinity for
OR3, followed by OR2 and OR1. cI has the
opposite affinity it prefers OR1, then OR2 and
OR3. When cI binds at OR1, it prevents RNA Pol
from binding at PR. Similarly, Cro binding at
OR3 blocks PRM. Therefore, Cro and cI can
compete for these binding sites. At lower cI,
Cro is able to outcompete cI at OR and OL and
stimulate lysis. At medium levels of Cro, PL
and PRM are disabled, but PR remains active,
producing O and P for lysis.
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Lysis 2
PR
STOP
cII
O
P
Cro
int
xis
cIII
N
cI
STOP
Cro
Cro
PR
PRM
OR3
OR2
OR1
PRM
PL
Cro
Cro
Cro
Products (None)
When Cro becomes high, all promoters are shut
off.
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Decision Time
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
PL
Products N, cIII, xis, int, Cro, cII, O, P
Now lets see what happens when lysogeny is
chosen.
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Lysogeny
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
PI
PRE
PRM
cII
cII
PL
cIII
cIII
Products N, cIII, xis, int, Cro, cII, O, P, cI.
Glucose was scarce, so cAMP was not exported and
cAMP is high. cAMP binds HflA, so HflA does
not degrade cII. The cIII protein helps protect
cII, and together they activate PI and PRE. This
results in more int protein, and the cI protein
(The l Repressor). The PRE promoter that leads to
production of cI is a very weak promoter. cII is
needed so that RNA Polymerase is able to bind
this promoter effectively. The transcript has a
Shine-Dalgarno sequence, so it is efficiently
translated once it is produced. This helps
accelerate initial production of cI. The PRM
promoter, which also produces cI, is not yet
active. When it is active, it is not efficiently
translated (about 8X less than PRE) because it
lacks the Shine-Dalgarno sequence. This promoter
is only needed for occasional production of cI
(in maintenance mode), so this reduced level of
expression is desirable.
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Lysogeny 2
cI
PR
STOP
cII
O
P
Cro
int
xis
cIII
N
cI
PRM
STOP
cI
PI
PR
OR3
OR2
OR1
PRM
PL
cI
cI
Products cI
At this point, enough proteins for integration
into the host genome have been produced, so
lysogeny begins. When cI is produced, it shuts
off all promoters except PM and maintains
lysogeny. It upregulates PM, until cI becomes
very high, where it then shuts off PM also.
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Induction
RecA
cI
PR
STOP
cII
O
P
Cro
int
xis
cIII
N
cI
PRM
STOP
cI
PR
OR3
OR2
OR1
PRM
PL
cI
cI
RecA
Products cI
RecA
RecA
When the host cell is in danger, its S.O.S.
response activates the hosts multi-purpose RecA
protein. RecA interacts with cI, and cI is
cleaved.
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Induction 2
PR
N
Cro
cII
O
P
int
xis
cIII
N
cI
N
Cro
Cro
PR
PRM
OR3
OR2
OR1
PRM
PL
Cro
Cro
Products Cro, cII, O, P
With RecA degrading any cI that is produced, Cro
is able to outcompete cI and begin lysis.
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References

• Tapprich, W. University of Nebraska Omaha.
  (2004) Virology notes.
• Cox, G. S. University of Nebraska Medical
  Center. (2006) BRTP 822 handouts.
• Echols, H., Murialdo, H. (1978) Microbiology
  Reviews 42577-591.