Lambda Phage

Infection

Bacteriophage Lambda binds to the target E. coli cell, the J protein in the tail tip interacting with the
lamB gene product of E. coli, a porin molecule which is part of the maltose operon.

The linear phage genome is injected past the cell outer membrane.

The DNA passes through a separate sugar transport protein (ptsG) in the inner membrane, and
immediately circularises using the cos sites, 12-base G-C rich cohesive "sticky ends". The single-
stranded nicks are ligated by host DNA ligase.

Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T rich regions to
unwind and drive transcription.

Transcription starts from the constitutive PL, PR and PR' promoters producing the 'immediate early'
transcripts. Initially these express the N and cro genes, producing N, Cro and a short inactive protein.

Cro binds to OR3 preventing access to the PRM promoter preventing expression of the cI gene. N
binds to the two Nut (N utilisation) sites, one in the N gene in the PL reading frame, and one in the
cro gene in the PR reading frame.

The N protein is an antiterminator, and functions to extend the reading frames that it is bound to.
When RNA polymerase transcribes these regions, it recruits the N and forms a complex with several
host Nus proteins. This complex skips through most termination codons. The extended transcripts
(the 'late early' transcripts) include the N and cro genes along with cII and cIII genes, and xis, int, OP
and Q genes discussed later.

The cIII protein acts to protect the cII protein from proteolysis by FtsH (a membrane-bound essential
E. coli protease) by acting as a competitive inhibitor. This inhibition can induce a bacteriostatic state,
which favours lysogeny. cIII also directly stabilises the cII protein[2]. On initial infection, the stability
of cII determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if cII
is degraded the phage will go into the lytic pathway. Low temperature, starvation of the cells and
high multiplicity of infection (MOI) are known to favor lysogeny (see later discussion).

Lytic Lifestyle

This is the lifecycle that the phage follows following most infections, where the cII protein does not
reach a high enough concentration due to degradation, so does not activate its promoters.

The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the
lambda genome (OP). Cro dominates the repressor site (see "Repressor"), repressing synthesis from
the PRM promoter.

The O and P proteins initiate replication of the phage chromosome (see "Lytic Replication").

Q, another antiterminator, binds to Qut sites.

Transcription from the PR' promoter can now extend to produce mRNA for the lysis and the head and
tail proteins.

Structural proteins and phage genomes self assemble into new phage particles.
Products of the lysis genes R and S, cause cell lysis at high enough concentrations. S is a holin which
makes holes in the membrane. R is an endolysin which cleaves the cell wall. Around 100 new phage
are released.

Rightward Transcription

Rightward transcription expresses the O, P and Q genes. O and P are responsible for initiating
replication, and Q is another antiterminator which allows the expression of head, tail and lysis genes
from PR’.

Lytic Replication

For the first few replication cycles, the lambda genome undergoes θ replication (circle-to-circle).

This is initiated at the ori site located in the O gene. O protein binds the ori site, and P protein binds
the DnaB subunit of the host replication machinery as well as binding O. This effectively
commandeers the host DNA polymerase.

Soon, the phage switches to a rolling-circle type of replication similar to that used by phage M13. The
DNA is nicked and the 3’ end serves as a primer. Notably, this doesn’t release single copies of the
phage genome, but rather one long molecule with many copies of the genome: a concatemer.

These concatemers are cleaved at their cos sites as they are packaged. Packaging cannot occur from
circular phage DNA, only from concatomeric DNA.

Q Antitermination

Q is similar to N in its effect: Q binds to RNA polymerase in Qut sites and the resulting complex can
ignore terminators, however the mechanism is very different.

The Qut site is very close to the PR’ promoter, close enough that the σ factor has not been released
from the RNA polymerase holoenzyme. The Qut site resembles the -10 Pribnow box, causing the
holoenzyme to pause.

Q protein then binds and displaces part of the σ factor and transcription re-initiates.

The head and tail proteins are transcribed and self-assemble.

Leftward Transcription

Leftward transcription expresses the gam, red, xis and int genes. Gam and red proteins are involved
in recombination. Gam is also important in that it inhibits the host RecBCD nuclease from degrading
the 3’ ends in rolling circle replication. Int and xis are integration and excision proteins which are vital
to lysogeny.

xis and int regulation of insertion and excision

xis and int are found on the same piece of mRNA, so approximately equal concentrations of xis and
int proteins are produced. This results (initially) in the excision of any inserted genomes from the
host genome.
The mRNA from the PL promoter forms a stable secondary structure with a bobby pin loop in the sib
section of the mRNA. This targets the 3' (sib) end of the mRNA for RNAaseIII degradation, which
results in a lower effective concentration of int mRNA than xis mRNA (as the int cistron is nearer to
the sib sequence than the xis cistron is to the sib sequence), so a higher concentrations of xis than int
is observed.

Higher concentrations of xis than int result in no insertion or excision of phage genomes, the
evolutionarily favoured action - leaving any pre-insterted phage genomes inserted (so reducing
competition) and preventing the insertion of the phage genome into the genome of a doomed host.

Lysogenic (or Lysenogenic) Lifestyle

This is the lifecycle that the phage follows after a small number of infections in specific conditions,
where the cII protein reaches a high enough concentration due to stabilisation and lack of
degradation, and so activates its promoters.

The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the
lambda genome.

The stablized cII acts to promote transcription from the PRE, PI and Pantiq promoters.

The Pantiq promoter produces antisense mRNA to the Q gene message of the PR promoter
transcript, thereby switching off Q production. The PRE promoter produces antisense mRNA to the
cro section of the PR promoter transcript, turning down cro production, and has a transcript of the cI
gene. This is expressed, turning on cI repressor production. The PI promoter expresses the int gene,
resulting in high concentrations of int protein. This int protein integrates the phage DNA into the host
chromosome (see "Prophage Integration").

No Q results in no extension of the PR' promoter's reading frame, so no lytic or structural proteins
are made. Elevated levels of int (much higher than that of xis) result in the insertion of the lambda
genome into the hosts genome (see diagram). Production of cI leads to the binding of cI to the OR1
and OR2 sites in the PR promoter, turning off cro and other early gene expression. cI also binds to the
PL promoter, turning off transcription there too.

Lack of cro leaves the OR3 site unbound, so transcription from the PRM promoter may occur,
maintaining levels of cI.

Lack of transcription from the PL and PR promoters leads to no further production of cII and cIII.

As cII and cIII concentrations decrease, transcription from the Pantiq, PRE and PI stop being
promoted since they are no longer needed.

Only the PRM and PR' promoters are left active, the former producing cI protein and the latter a
short inactive transcript. The genome remains inserted into the host genome in a dormant state.

Prophage Integration

The integration of phage λ takes place at a special attachment sites in the bacterial and phage
genomes, called attλ. The sequence of the bacterial att site is called attB, between the gal and bio
operons, and consists of the parts B-O-B', whereas the complementary sequence in the circular
phage genome is called attP and consists of the parts P-O-P'. The integration itself is a sequential
exchange (see genetic recombination) via a Holliday junction and requires both the phage protein Int
and the bacterial protein IHF (integration host factor). Both Int and IHF bind to attP and form an
intasome, a DNA-protein-complex designed for site-specific recombination of the phage and host
DNA. The original B-O-B' sequence is changed by the integration to B-O-P'-phage DNA-P-O-B'. The
phage DNA is now part of the host's genome.

Maintenance of Lysogeny

Lysogeny is maintained solely by cI. cI represses transcription from PL and PR while upregulating and
controlling its own expression from PRM. It is therefore the only protein expressed by lysogenic
phage.

This is coordinated by the PL and PR operators. Both operators have three binding sites for cI: OL1,
OL2, and OL3 for PL, and OR1, OR2 and OR3 for PR.

cI binds most favorably to OR1; binding here inhibits transcription from PR. As cI easily dimerises, the
binding of cI to OR1 greatly increases the affinity of the binding of cI to OR2, and this happens almost
immediately after OR1 binding. This activates transcription in the other direction from PRM, as the N
terminal domain of cI on OR2 tightens the binding of RNA polymerase to PRM and hence cI
stimulates its own transcription. When it is present at a much higher concentration, it also binds to
OR3, inhibiting transcription from PRM, thus regulating its own levels in a negative feedback loop.

cI binding to the PL operator is very similar, except that it has no direct effect on cI transcription. As
an additional repression of its own expression, however, cI dimers bound to OR3 and OL3 bend the
DNA between them to tetramerise.

The presence of cI causes immunity to superinfection by other lambda phages, as it will inhibit their
PL and PR promoters.

Induction

The host cell, containing a dormant phage genome, experiences DNA damage due to a high stress
environment, and starts to undergo the SOS response.

RecA (a cellular protein) detects DNA damage and becomes activated. It is now RecA*, a highly
specific co-protease.

Normally RecA* binds LexA (a transcription repressor), activating LexA auto-protease activity,which
destroys LexA repressor allowing production of DNA repair proteins. In lysogenic cells this response is
hijacked, and RecA* stimulates cI autocleavage.

Cleaved cI can no longer dimerise, and loses its affinity for DNA binding.

The PR and PL promoters are no longer repressed and switch on, and the cell returns to the lytic
sequence of expression events (note that cII is not stable in cells undergoing the SOS response).
There is however one notable difference.

Control of phage genome excision in induction

The phage genome is still inserted in the host genome and needs excision for DNA replication to
occur. The sib section beyond the normal PL promoter transcript is, however, no longer included in
this reading frame (see diagram).

No sib domain on the PL promoter mRNA results in no hairpin loop on the 3' end, and the transcript
is no longer targeted for RNAaseIII degradation.

The new intact transcript has one copy of both xis and int, so approximately equal concentrations of
xis and int proteins are produced.

Equal concentrations of xis and int result in the excision of the inserted genome from the host
genome for replication and later phage production.

Genome Structure

Protein Function Overview

cro; (Control of Repressor's Operator) Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 >
OR2 = OR1, ie. preferentially binds OR3). At low concentrations blocks the pRM promoter (preventing
cI production). At high concentrations downregulates its own production through OR2 and OR1
binding. No cooperative binding (c.f. below for cI binding)

cI; (Clear 1) Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 = OR3, ie.
preferentially binds OR1). At low concentrations blocks the pR promoter (preventing cro production).
At high concentrations downregulates its own production through OR3 binding. Binding of cI at OR1
stimulates an almost simultaneous cI binding to OR2 via cooperative binding (via cI C terminal
domain interactions) N terminal domain of cI on OR2 tightens the binding of RNA polymerase to pRM
and hence stimulate its own transcription. Repressor also inhibits transcription from the pL
promoter. Susceptible to cleavage by RecA* in cells undergoing the SOS response.

cII; (Clear 2) Transcription activator. Activates transcription from the pAQ, pRE and pI promoters. Low
stability due to susceptibility to cellular HflB (FtsH) proteases (especially in healthy cells and cells
undergoing the SOS response.

cIII;(Clear 3) HflB (FtsH) binding protein, protects cII from degradation by proteases.

N; (aNtiterminator) RNA binding protein and RNA polymerase cofactor, binds RNA (at Nut sites) and
transfers onto the nascent RNApol that just transcribed the nut site. This RNApol modification
prevents its recognition of termination sites, so normal RNA polymerase termination signals are
ignored and RNA synthesis continues into distal phage genes.
Q; DNA binding protein and RNApol cofactor, binds DNA (at Qut sites) and transfers onto the
initiating RNApol. This RNApol modification alters its recognition of termination sequences, so
normal ones are ignored; special Q termination sequences some 20,000 bp away are effective.

xis; (eXcISion) excisionase and Int protein regulator, manages excision and insertion of phage genome
into the host's genome.

int; (INTegration) Int protein, manages insertion of phage genome into the host's genome. In
Conditions of low int concentration there is no effect. If xis is low in concentration and int high then
this leads to the insertion of the phage genome. If xis and int have high (and approximately equal)
concentrations this leads to the excision of phage genomes from the host's genome.

A, B, C, D, E, F, Z, U, V, G, T, H, M, L, K, I, J [Shown on diagram as head and tail, A-F code for phage

head genes, Z-J code for phage tail genes. The order shown here is as found on the genome, reading
in a clockwise direction]; structural proteins, self assemble with the phage genome into daughter
phage particles.

S, R [Shown on diagram as lysis. The order shown here is as found on the genome, reading in a
clockwise direction]; cause the host cell to undergo lysis at high enough concentrations.

OP [Shown on diagram as O replication P]; DNA replication functions, promotes the specific
replication of only the phage genome.

sib [not a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in
transcribed mRNA beyond int. Attracts degradation of mRNA by RNAaseIII.

attP [not a protein, but a conserved DNA sequence]; point of action of Int and Xis in integration and
excision of the phage genome into the host's genome. Corresponding attB found in the host's
genome at the point of insertion.

Repressor

The repressor found in the phage lambda is a notable example of the level of control possible over
gene expression by a very simple system. It forms a 'binary switch' with two genes under mutually
exclusive expression, as discovered by Barbara J. Meyer.[3]

The lambda repressor gene system consists of (from left to right on the chromosome):
cI gene

OR3

OR2

OR1

cro gene

The lambda repressor is a dimer also known as the cI protein. It regulates the transcription of the cI
protein and the Cro protein.

The life cycle of lambda phages is controlled by cI and Cro proteins. The lambda phage will remain in
the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if cro
proteins predominate.

The cI dimer may bind to any of three operators, OR1, OR2, and OR3, in the order OR1 > OR2 = OR3.
Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, an effect called
cooperativity. Thus, OR1 and OR2 are almost always simultaneously occupied by cI. However, this
does not increase the affinity between cI and OR3, which will be occupied only when the cI
concentration is high.

In the absence of cI proteins, the cro gene may be transcribed.

In the presence of cI proteins, only the cI gene may be transcribed.

At high concentration of cI, transcriptions of both genes are repressed.

Lytic or Lysogenic?

The gene regulatory circuitry of phage λ is among the best-understood circuits at the mechanistic
level. This circuitry involves several interesting regulatory behaviors. An infected cell undergoes a
decision between two alternative pathways, the lytic and lysogenic pathways. If the latter is followed,
the lysogenic state is established and maintained. While this state is highly stable, it can switch to the
lytic pathway in the process of prophage induction, which occurs when the host SOS response is
triggered by DNA damage.[4].

Simplistically, in cells with abundant nutrients, protease activity is high, which breaks down cII. This
leads to the lytic lifestyle. In cells with limited nutrients, protease activity is low, making cII stable.
This leads to the lysogenic lifestyle.