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Phage Lambda (λ)Comments

Phage Lambda (λ)

Bacteriophage lambda (λ) was discovered by Joshua and Esther Lederberg. While mutagenizing strains E. coli using UV, a strain was found to be a lysogen.

  • Genome is 48,502 bp dsDNA with 12 nucleotide ssDNA cohesive termini. Contained in capsid.
  • Linear dsDNA circularizes due to annealing between 3' sticky cos sites.
  • The capsid has two parts:
    1. Head, composed of B, C, Nu3, D, and E proteins.
    2. Tail, composed of J and H proteins.
  • Viral Receptor is the E. coli maltose receptor (product of lamB) on surface of host cell.
  • Temperate phage, meaning that it can undergo either lysogeny or lysis.
  • Chooses developmental pathway depending on nutrient availability.
  • Bacterophage λ has two different life cycles.
    • Lytic, where new phages are synthesized, which then lyse the host and burst from the cell.
    • Lysogenic, where the phage genome integrates into the host genome. It is replicated along with host genome host cell replication. Such a host is called a lysogen.
Int Pi tL N OL,PL cI PRM OR3 OR2 OR1 PR Cro PRE cII O P tr2 Q
red dot / / -------- green dot ----------- red dot ------ ------ ------ green dot red dot // -------
 
protein coding gene required for lytic replication
green dotpromoter active during lytic replication
protein coding gene required for lysogeny
red dotpromoter required for lysogeny
PRThe rightward promoter, transcribed to make Cro & extended by N to make lytic cycle genes O & P. OR3 overlaps PRM.
PLThe leftward promoter, transcribed by Pol to make N. Later extended by N to make the integrase gene for lysogeny.
HflA cellular protease cleaving cII. Function at high [glucose], promoting lysis.
NN is a viral protein that extends transcription from PR into O and P and from PLinto integrase gene. DNA binding protein and RNApol cofactor, binds DNA (at Nut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special N stop codons are effective instead.
cIIMade from PR promoter, cII activates PRE to make cI and activates Pi to make integrase. In this way, cII promotes lysogeny. cII outcompetes Cro, unless Hfl protease degrades cII (this only happens when there is high [glucose]. cII is unstable due to succeptability to cellular proteases (especially in healthy cells and cells undergoing the SOS response), slightly stabilised by binding to cIII.
CroMade from PR promoter, Cro represses transcription from PRM by binding to OR3. At high concentrations it binds to OR2 & OR1, thereby blocking Pol from binding to PR. Cro is a repressor just like cII, but it binds the operators (like OR3) with the opposite affinity from cI. When Cro binds OR3 it represses transcription of cI gene from PRM. RNAP is already trancribing CRO from PR as well as O and P genes with help from N. At higher levels, CRO also autoregulates itself by binding to OR2 and OR1 and repressing transcription from PR. Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 > OR2 > OR1, ie. prefferentially binds OR3). At low concentrations blocks the R promoter (preventing cI production). At high concentrations downregulates its own production through OR2 and OR1 binding.
cIMade by activation of PRE promoter through action of cII on PRE, cI is an activator of PRM, recruiting RNAP to OR3 by binding cooperatively to OR1 and OR2. Simultaneously represses PR, occupying RNAP site near OR1. At high [cI], binds to OR3 to block polymerase's ability to transcribe cI gene, shutting itself off. Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 > OR3, ie. prefferentially binds OR1). At low concentrations blocks the RM promoter (preventing cro production). At high concentrations downregulates its own production through OR2 and OR3 binding. Also inhibits transcription from the L promoter. Succeptable to cleavage by RecA* in cells undergoing the SOS response.
cIIIcII binding protein, protects cII from degradation by cellular proteases.
PR & PLStrong promoters. The other promoters must be activated. PRE is the promoter for repressor establishment.
QDNA binding protein and RNApol cofactor, binds DNA (at Qut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special Q stop codons are effective instead.
xisexcisionase and integrase regulator, manages excision and insertion of phage genome into the host's genome.
intintegrase, 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 the n 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-F code for phage head genes.
Z-JZ-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, RLysis promoters, cause the host cell to undergo lysis at high enough concentrations.
OP [Shown on diagram as O replication P]; DNA replication promoter, promotes the specific replication of only the phage genome.
SIBNot a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in transcribed mRNA. Attracts degradation of mRNA by RNAaseIII.
attpattP not a protein, but a vital conserved DNA sequence]; point of action of int and xis in insertion and excision of the phage genome into the host's genome. Corresponding attb found in the host's genome at the point of insertion.
Lifecycle
Infection
  • Bacteriophage λ tail fibers bind to E. coli maltose receptors.
  • The linear phage genome is injected and immediately circularizes.
  • Transcriptions starts from the L, R, and R' promoters to produce N, Cro, and a short inactive protein (all immediate early products).
  • Cro binds to OR1, preventing access to Repressor Maintenance Promoter (RMP) and preventing trancription of cI.
  • N binds to the two nut sites, one on N and one on Cro.
  • The N bound in the L and R ORFs extends the reading grames. Early translation of these (the 'late early' transcripts) are more N and Cro along with cII and cIII.
  • cIII binds to cII partially preventing protease vulnerability. The stability of cII determines the lifestyle of the phage. In unstressed cells with abundant nutrients protease activity is high, and cII unstable. This leads to the lytic lifestyle. In stressed cells with limited nutrients protease activity is low, and cII stable. This leads to the lysogenic lifestyle.
Immediately after infection
  • pL and pR promote transcription of N and Cro.
  • N prevents RNAP from terminating, so transcription continues.
  • This continued translation results in cII (lysogenic) and Cro (lytic) synthesis.
  • At this point, there are 2 possibilities:
    • Intracellular [glucose] is low, cII accumulates, outcompeting Cro, and lysogeny ensues.
    • Intracellular [glucose] is high, cII is degraded, Cro is dominant, and lytic cycle ensues.
N-Antitermination Mechanism Details

Read this section after you understand lysogeny and the lytic cycle)

  • This probably evolved to prevent expression of replication and other lytic functions (especially cell lysis proteins) in a lysogen.
  • cI represss txn from PR about 1000x. However, since PR is so strong there is a low level of transccription from PR in presence of cI. In lysogen, these transcripts mostly terminate at tR1 so that insufficient O & P proteins are made to initiate replication from λ replication origin in prophage. Virtually all transcripts from PR are terminated at tR2 so that no Q protein is made in a lysogen, and hence no lytic genes are expressed. Since cI also repressed pL, insufficient N protein is made to anti-terminate transcripts from PR.
  • In absence of other viral proteins, txn from PR terminates at tR to the right of the Cro gene.
  • Transcription from PL terminates at tL to the left of the N gene.
  • N protein expressed from PL acts to prevent termination at tR and tL
  • N is an antiterminator
  • N functions by binding to specific RNA sequences called nut sites for N utilization.
  • N binds to RNA with the nut site sequence.
  • The nut site contains a stem and a loop.
  • N binds immediately to the nut sites immediately after its synthesis.
  • N binds together with several host cell proteins called Nus proteins.
  • Host cell Nus proteins function during transcription of rRNA genes to prevent RNAP from terminating txn.
  • Without Nus proteins, RNAP is unable to txn through a rRNA gene because of multiple termination signals resulting from extensive 2º structure of rRNA.
  • The complex of N and host cell NUs proteins then binds to the RNAP and prevents termination.
  • Diagram binding of N to nut site and RNAP and continued txn by N.
  • N anti-termination results in txn of cII, and O and P, and Q, all genes required for lytic cycle.
  • Q is required for transcription of the late genes.
Lytic vs. Lysogenic Decision Details

Read this section after you understand lysogeny and the lytic cycle)

  • cII is extremely unstable.
  • It is degraded by host cell protease Hfl (mutants in Hfl have much more stable cII relative to WT and ∴ almost always undergo lysogeny when infected by λ).
  • Hfl is regulated by [cAMP].
  • When [glucose] is high, [cAMP] is low. This leads to high Hfl activity and rapid degradation of cII.
  • This prevents expression of cI from PRE, so the cell undergoes lytic pathway.
  • Intracellular [glucose] is high, ∴ [cAMP] is low ∴ Hfl has high activity and cleaves cII. As a result, Cro is dominant and lytic cycle ensues.
  • When [glucose] is low, adenyl cyclase is activated and [cAMP] is high, resulting in low Hfl protease activity.
  • Consequently, cII is stabilized and txn from PRE and PI is activited, leading to lysogeny
  • Intracellular [glucose] is low, ∴ [cAMP] is high ∴ Hfl has low activity and does not cleave cII. As a result, cII accumulates, outcompeting Cro, and lysogeny ensues.
  • By this mechanism, λ replicates in host cells with sufficient nutrients to produce large numbers of progeny, but to follow lysogeny when host cells lack enough nutrients to produce progeny virions.
Lysogenic Cycle (aka Lysenogenic Cycle)
  • cII functions, and it activates PRE and Pi, thereby producing cI and large amounts of Integrase.
  • cI has 4 important functions
    1. cI binds to OR1 and OR2, recruiting RNAP to PRM and repressing PR. Without cI, RNAP does not bind to pRM. cI simultaneously interacts with OR3 and σ70 subunit of RNAP.
    2. cI binds to OL1 and OL2, repressing PL (preventing synthesis of N)
    3. At very high [cI], cI will bind to OR3 and thereby repress its own production (it autoregulates itself)
    4. In addition, cI inhibits replication of super-infecting phage by repressing PR & PL.
  • During lysogeny, 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome. Although the stable cII also acts to promote transcription from the RE, I and antiq promoters.
  • The antiq promoter produces antisense mRNA to the Q section of the R promoter transcript switching off Q production.
  • No Q results in no extension of the R' promoter's reading frame, so no lytic or structural genes are made.
  • Elevated levels of integrase (to 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 OR3 site in the R promoter, turning off cro production. cI also binds to the L promoter, turning off transcription there too.
  • Lack of cro leaves the OR1 site is left unbound, so transcription from the RM promoter may occur, maintaining levels of cI.
  • Lack of transcription from the L and R promoters leads to no further production of cII and cIII.
  • As cII and cIII concentrations decrease, transcription from the antiq, RE and I stop being promoted.
  • Only the RM and R' promoters are left active, producing a short inactive transcript and cI. The genome is inserted in the host and is in a dormant state.
Integration
  • Site-specific recombination by λ integrase + Excisase leads to excision of λ DNA. Immediately after cleave of cI during prophage induction, then N is synthesized and prevents termination by RNAP that initiated at pR and pL.
  • To promote integration, lambda wants to makie the int protein and not the xis protin. To promote integration, the mRNA transcribed from Pi has an ATG for translation of the into protein, not xis. During lytic phase, lambda wants to favor viral DNA replication, not integration. It makes an unstable mRNA from PL, and degrades it from the 3' end. This results in little protein, and more xis than int. For induction out of lysogeny, lambda wants an equal amount of int and xis protein. It there puts sib on the other side of the integration site in the chromosome, thus preventing the sib structure from forming. Gel shift assay or EMSA.
  • To promote integration, Bacteriophage λ wants to make the int protein and not xis protein. mRNA transcribed from Pi has an ATG for translation of int protein, not xis. The integration of phage λ takes place at a special attachment site in the bacterial genome, called attλ. The sequence of the att site is called attB 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 structure and requires both the phage protein int and the bacterial protein IHF (integration host factor). Both int and IHF bind to attP and built an intrasome, a DNA-protein-complex designed for site-specific recombination of the phage and host DNA. The original BOB' secuanes 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.
Lytic Cycle
  • Little cI is made, so:
    • No transcription from PRE nor Pi, so no cI nor Integrase is expressed.
    • Transcription from PR and PL is not repressed.
  • Expresses of cro represses prm, repressing lysogeny.
    • Cro is a repressor binding to operators with opposite affinity from cI. Cro binds OR3 and repressing transcription of cI from PRM.
    • cII favors lysogeny by activating transcription of the cI gene from PRE.
    • cII outcompetes Cro unless cII is degraded by Hfl.
    • When cI binds to OR1 and OR2 it simultaneously recruits the RNAP to PRM and blocks synthesis from PR.
    • At higher levels, Cro also autoregulates itself by binding to OR2 and OR1 and repressing its own transcription from PR.
  • N is made from the transcript from PL.
  • N is made from the transcript from PL.
  • N antiterminates, allowing genes to right of PR, O and P for viral DNA replication and Q for transcription of late genes from Late promoter, to be transcribed.
  • O and P causes initiation of DNA replicaiton by host cell enzymes.
  • Q activates expression of late genes.
  • Lytic replication ensues.
Prophage induction.

When a high stress environment results in DNA damage, the cell performs excision repair of DNA:

  • Damaged DNA is removed, resulting in a gap of ssDNA.
  • DNAP fills in the gap.
  • DNA Ligase repairs any nicks in the newly formed DNA.

In addition, cells with damaged DNA undergo the SOS response.

  • RecA, a host protein, detects DNA damage and becomes RecA*, an activated highly specific protease.
  • LexA is a dimer that represses transcription of sos genes (which help the cell cope with DNA damage), and is cleaved by RecA*.
  • The dimer, consisted of two monomers, is held together between C-terminal domains.
  • The DNA-binding domain of LexA, once cleaved, no longer binds to sos genes, derepressing sos genes.
  • In infected cells, RecA* cleaves cI because cI has a structure very similar to LexA.
  • Cleaved cI loses its binding affinity to DNA, resulting in PR and PL no longer being repressed.
  • cII is not stable in cells undergoing the SOS response.

λ exploits a host cell system that regulates expression of SOS genes.

  • RecA, activated by binding to ssDNA, cleave a cellular repressor called LexA. This derepresses the SOS genes.
  • cI evolved to be sensitive to digestion by activated RecA, so that λ prophage are induced when host cell DNA is damaged.
  • Damage to cellular DNA leads to exposure of ssDNA.
  • RecA protein binds to the exposed ssDNA.
  • This activates a protease that cleaves cI.
  • Cleaved cI cannot repress PR or PL, consequently, early λ transcription begins.
  • cI is a dimer. Most of the monomer-monomer interaction that holds it together is between the C-terminal domains.
  • Activated RecA cleaves cI in the linker region. The cleaved N-terminal DNA-binding domain has too low an affinity to bind to OR or OL.
Summary
  • cII is expressed from PR during early stages of infection, when RNAP is antiterminatd by N protein.
  • cII activates transcription from PRE, the promoter for establishment of lysogeny.
  • cII binds to PRE cooperatively with RNAP, activating transcription to the left from PRE.
  • cII and RNAP bind to the PRE promoter cooperatively, so that cII strongly activates transcription from PRE.
  • cI can be translated from the mRNA transcribed from PRE.
  • If cI is made in larger amounts than Cro, then cI repressed PR and PL, favoring lysogeny.
  • cII also activates transcription from PI, the promoter for integrase expression.
  • Integrase is translated, causing integration of lambda DNA.
  • Integrase is only made transiently, because cII is only made transiently.
  • Once cII has activated expression of cI, cI represses transcription from PR and cII expression is shut off.
  • However, once cI is expressed from transcript from PRE, cI maintains expression by autoregulating transcription form PRM.
  • Lytic pathway is followed when cII activity is so low that cI and Integrase are not expressed early after infection. Transcription from PR and PL is maintained, and regulated to proper level by Cro synthesis which represses txn from these promoters when it reaches too high a level. Cro expression also repressed txn from PRM, ensuring that the decision to follow the lytic pathway is irreversible.

The 'late early' transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
The lambda genome is replicated in preparation for daughter phage production.
Q binds to Qut sites.
Replication from the R' promoter can now extend to produce mRNA for the lysis and the structural proteins.
Structural proteins and phage genomes self assemble into new phage particles.
Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape.

[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 L promoter forms a stable secondary structure with a hairpin loop in the sib section of the mRNA. This targets the 5' end of the mRNA for RNAaseIII degradation, so a lower effective concentration of xis mRNA than int mRNA is found, so higher concentrations of xis than int.
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.

The lambda repressor is a dimer also known as the cI protein. It regulates the transcription of the cI protein and the Cro protein. cI and Cro proteins regulate λ life cycle. If cI predominates, the lysogenic cycle will ensure. If Cro proteins predominate, the lytic cycle will ensue. cI dimer binds to 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.

How does lambda replicate and package viral DNA? Nu1/A (terminase) binds cos sites.