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MCB3026F > Lambda Phage > Flashcards

Lambda Phage Flashcards

1
Q

Describe the lytic lifecycle of a phage

A
  1. Adsorption & penetration: Phage attaches to specific receptor and injects dsDNA
  2. Phage forces bacteria to make virus DNA & proteins
    a. Early mRNA made
    b. Host DNA degraded
    c. Phage DNA made
    d. Late RNA made
    e. Heads and tails made
    f. Heads filled
  3. Phages assembled where virus DNA packed within virus protein coat (virions formed)
  4. Mature virus released by cell lysis
  5. Phages that reproduce using a lytic cycle = virulent phages
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2
Q

Describe the lysogenic lifecycle of a phage

A
  1. Adsorption and penetration
  2. Phage doesn’t take control of bacterium: Virus genome integrates into host genome = Prophage
  3. Phage DNA reproduced along with bacterial chromosome for long periods & appear normal
    a. Bacteria + integrated phage = Lysogens
  4. Lysogens can produce phage particles under DNA damage conditions
    a. i.e. when gets difficult phage can leave bacterial genome – enter lytic cycle and leave
  5. Phages that can establish lysogeny = temperate phages
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3
Q

What is a prophage?

A

Virus genome integrated into host genome is now called a prophage

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4
Q

What is a lysogen?

A

Bacteria + integrated phage

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5
Q

What is the structure of bacteriophage Lambda?

A
  • Head/capsid (has ds linear genome)
  • Collar
  • Tail
  • Tail fiber
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6
Q

Specificity of the Lambda Phage

A

Specifically attacks E. coli

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7
Q

Steps in Lambda Phage entry

A
  1. Adsorption
    a. Phage identifies host E. coli
    b. J protein in the tail tip binds to outer membrane protein (LamB)
  2. Phage DNA Injection
    a. Linear phage genome ejected into cell via E. coli sugar transport protein (PstM) located in inner membrane
  3. DNA Circularization and Ligation
    a. Phage DNA circularizes using cos sites (12bp GF rich cohesive ends)
    b. Host DNA ligase seals nicks – prevents exonuclease degradation
  4. Supercoiling
    a. Host DNA gyrase supercoils lambda DNA – AT rich regions unwind and drives transcription
  5. Theta Replication
    a. Phage replicates initially via Theta replication producing copies of the phage
    b. Transcription & translation of certain phage genes
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8
Q

PL - Name Derivation & Function

A

Promoter Leftward

  • Promoter for transcription of N, cIII, xis and int genes;
  • Important in establishing lysogeny
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9
Q

OL - Name Derivation & Function

A

Operator Leftward

  • Binding site for lambda repressor and Cro protein
  • Binding by lambda repressor maintains lysogenic state
  • Binding by Cro protein prevents establishment of lysogeny
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10
Q

PR - Name Derivation & Function

A

Promoter Rightward

  • Promoter for transcription of cro, cII, O, P and Q genes
  • Cro, O, P and Q proteins are needed for lytic cycle
  • CII protein helps establish lysogeny
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11
Q

OR - Name Derivation & Function

A

Operator Rightward

  • Binding site for lambda repressor and Cro protein
  • Binding by lambda repressor maintains lysogenic state
  • Binding by Cro allows transcription to occur
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12
Q

PRE - Name Derivation & Function

A

Promoter for Lambda Repressor Establishment

  • Promoter for cI gene (lambda repressor gene)
  • Recognized by CII protein, a transcriptional activator
  • Important in establishing lysogeny
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13
Q

PI - Name Derivation & Function

A

Promoter for Integrase Gene

  • Transcription from PI generates mRNA for integrase protein but not excisionase
  • Recognized by the transcriptional activator CII
  • Important for establishing lysogeny
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14
Q

PAQ - Name Derivation & Function

A

Promoter for Anti-Q mRNA

  • Transcription from PAQ generates an antisense RNA that binds Q mRNA, preventing its translation
  • Recognized by the transcriptional activator CII
  • Important for establishing lysogeny
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15
Q

PRM - Name Derivation & Function

A

Promoter for Repressor maintenance

  • Promoter for transcript of lambda repressor gene (cI)
  • Activated by lambda repressor
  • Important in maintain lysogeny
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16
Q

PR’ - Name Derivation & Function

A

Promoter Rightward’

  • Promoter for transcription of viral structural genes
  • Activated by Q protein
  • Important in lytic cycle
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17
Q

POOP - Name Derivation & Function

A

Promoter for Origin O & P

  • Transcription from POOP generates OOP antisense RNA that binds to the 3’ end of CII mRNA and intergenic space.
  • It is repressed by host LexA.
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18
Q

Phases of Lambda phage Transcription

A

Phase 1 = Immediate Early Transcription
Phase 2 = Delayed Early Transcription
Phase 3 = Late Lytic OR Late Lysogenic Transcription

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19
Q

Lambda Immediate Early Transcription

A

o Leftward:
• RNA polymerase binds at PL (Promoter Leftward) -> N protein (anti-terminator protein)
v Polymerase transcribes left and stops at TL (Terminator Leftward)

o Rightward:
• RNA polymerase binds at PR (Promoter Rightward) -> Cro Protein (anti-repressor) – acts in lysis
• Polymerase transcribes till it reaches TRI (Terminator Rightward I)

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20
Q

Lambda Delayed Early Transcription

A
  • N protein acts as an antiterminator to extend transcripts from PL and PR
  • RNA polymerase able to transcribe passed TL and even past TI (Terminator Integrase) and past TRI and TRII.
  • N protein acts to delay the transcription of these other proteins- N protein has to be transcribed first.
-	These proteins are - LEFT:
	o	CIII = Lysogeny
	o	Int & Xis = Integration/Excision
-	These proteins are - RIGHT:
	o	CII = Lysogeny
	o	O&P = ReplicatN
	o	Q = Lysis
  • CII Transcriptional Activator responsible for producing CI
  • Lysis - Lysogeny Decision made
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21
Q

What is the mechanism of anti-termination by the N protein?

A
  • N protein is unfolded (metabolically unstable) & binds to box B sequence in nutL & nutR sites on mRNA transcript (Just before tL & tR1 – see diagram)
  • Box B forms a hairpin – would usually terminate transcription – recognized by N protein
  • Binds with 4 different host bacterial proteins (Nus proteins) & RNA Pol
  • Allows RNA Pol to read-thru terminators tL/ti and tR1/tR2
    o i.e. skips the terminator hairpin
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22
Q

What are the advantages of retrogregulation at the Sib site?

A
  1. Avoiding competition if other phage is already in host genome
  2. Avoiding committing to lifecycle before it knows the environmental factors
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23
Q

Process of Retroregulation at the Sib site

A
  • To prevent the phage from having to decide between lysis and lysogeny yet – doesn’t want too much int or xis – because it hasn’t yet decided.
  • After RNA Pol passes ti it reaches the sib site
  • The sib site forms a hairpin loop which is recognized by RNAse III and cleaved
  • Host exonucleases then degrade the free 3’ end
  • Because integrase is 3’ with reference to exicionase – more integrase is degraded than excisionase
  • Resulting in less [Int] and [Xis]
    o i.e. ALWAYS less int than Xis
24
Q

Why is retroregulation required?

A
  • For integration to occur – requires excess Int
  • Excision only requires equal amounts of Int & Xis
  • If Int & Xis are expressed:
    • Are on the same mRNA, so if translated will lead to Int & Xis in equal conc. = Possibility of excision
  • When Sib retroregulation occurs:
    • Int closer to Sib on mRNA transcript and degrades faster than Xis therefore:
    • [Int] < [Xis] = no insertion or excision of phage
25
Q

What is CII a transcriptional activator of?

A

Pi, PRE and PAQ (Promoter Integrase, Promoter for Repressor Establishment, Promoter for Antisense Q RNA)

o These promoters cannot be activated (RNA Pol can’t bind) if CII isn’t bound there

26
Q

What does CII binding depend on? i.e. sequence specificity

A
  • CII binding depends on TTGC Sequence (flank the -35 consensus sequence)
    o Lambda phage DOESN’T have a well conserved -35 consensus sequence (not a lot of homology)
    o BUT the flanking TTGC sequence are FULLY conserved amongst Lambda phages & any mutation in this sequence = CII can’t bind & phage can’t establish lysogeny = forced into lysis
  • CII + PRE = CI repressor (lambda repressor) + it is also antisense RNA to Cro mRNA
  • CII + PRM = CI repressor
  • CII + PI = Integrase – allows you to override Sib retroregulation
  • CII + PAQ = antisense RNA to Q mRNA (Q involved in establishing lysis – makes head and tail. SO this prevents lysis from occurring)
27
Q

What is the purpose of CIII?

A

CIII partially protects CII from degradation

28
Q

What determines the decision between lysis or lysogeny?

A

A race between Pro protein and CI binding at the OR cis site

29
Q

What is the Cro OR binding affinity?

A

Cro protein binds OR3 1st, then OR2 then OR1 as [Cro] increases

30
Q

What is the CI OR binding affinity?

A

CI repressor binds OR1 1st, then OR2, then OR3 as [CI] increases

31
Q

Mechanism of lysis/lysogeny decision

A
  • CI initially made from PRE:
    o CI prevents transcription from PR when bound to OR1 and OR2
    o When phage integrated (prophage) CI allows transcription from PRM
    o Increased CI binds OR3 – prevents transcription from PRM
    o CI leads to lysogeny
  • Cro made from PR:
    o Cro binding to OR3 inhibits PRM but still allows transcription from PR
    o Increased Cro binds to OR2 and OR1 and prevents transcription from PRM and PR
    o Cro leads to lytic cycle
  • Lysogeny:
    o Increased [CI] in cell than [Cro]
    o CI binds to OR1 then OR2 – stimulating transcription of PRM (Increases CI) and decreasing PR transcription (no Cro)
    o Once confirmed that decision is lysogeny – CI will increase enough to bind OR3 = stops transcription from both promoters (PR & PRM)
  • Lysis:
    o Higher [Cro] in cell than [CI]
    o Cro binds OR3 – prevents transcription from PRM & stims transcription from PR = Increased [Cro] (decided on lysis)
    o Cro increases enough to binds OR2 and OR1 – prevents transcription of both promoters
32
Q

What biochemical markers are involved in assessing the environmental status of a host bacterial cell?

A

Proteases, RNAse levels, cAMP levels

33
Q

What protease is of interest in the lambda phage host bacterium?

A

FtsH protease of the bacterial (HflBCK) protease complex

34
Q

Why does FtsH degrade proteins?

A
  1. Quality control of aberrant/abnormal proteins

2. Regulated proteolysis of intact proteins under specific conditions

35
Q

What is FtsH protease activity dependant on?

A 
1.	Alarmone levels: 
	o	Increased (p)ppGpp = Decreased FtsH activity
  1. cAMP levels:
    o High cAMP indicates low glucose levels
    o cAMP negatively regulates FtsH protease activity
36
Q

Which RNAse is involved in degraded CII mRNA in lambda phage?

A

RNAse III

37
Q

How does RNAse III reflect host cell environmental status? And what is its effect on the Lysis/Lysogeny decision?

A

RNase III levels in the cell (generally increased growth of cell = increased RNAse III levels):

a. Increase and decrease with cell growth rate - reflecting growth rate regulation of RNAse III expression itself
b. Activates N gene translation by ±200 fold
c. Promotes degradation of the CII mRNA
38
Q

What is the effect of FtsH protease, RNAse III, and CIII on the lysis/lysogeny decision?

A
  1. FtsH protease degrade CII & CIII
  2. RNAse III degrades CII mRNA
  3. CIII protects CII from degradation by acting as a substrate & competitive inhibitor of FtsH

Therefore - CII and CIII is used to assess environmental status via FtsH and RNase III levels.

39
Q

How does a lambda phage assess which lifecycle to enter based on environmental cues?

A

Good Environment:

  • Nutrient rich, High [glucose], Active Growth, AA available, High rate DNA replication
  • High Growth
  • High [FtsH] & [RNAse III] - Low [alarmone] & [cAMP]
  • Low [CII] + insufficient CIII protection
  • CI not transcribed = Cro wins race at OR = lytic cycle

Poor Environment:

  • Nutrient poor, Low [glucose], Slow Growth, Limited AA, Low rate DNA replication
  • Poor Growth
  • Low [FtsH] & [RNAse III] - High [alarmone] & [cAMP]
  • High [CII] + sufficient CIII protection
  • CI actively transcribed = CI wins race at OR = Lysogenic cycle
40
Q

How does Alarmone regulate the Lysis/Lysogeny decision?

A

(p) ppGpp affects:
1. FtsH protease activity:
a. RelA/SpoT control FtsH levels
b. A modest increase in Alarmone leads to lower levels of FtsH

2. Modulation of transcription of Lambda phage promoters:
	a. Modest increase in ppGpp levels also increases expression of pR, pRE and pI
41
Q

The Late Lytic Pathway

A
  1. High [FtsH, RNase III] = Cro wins the race
  2. Cro binds to OR – stops transcription from PRM – stims PR transcription
    o No N protein, no CII/CIII = no CI (No lysogeny)
    o Transcribes more Cro, and O, P, Q
    o O & P involved in phage replication – switches from theta replication to rolling circle
  3. Enough Q protein transcribed to act as anti-terminator by binding at qut site on the DNA
    o Binds to qut site between -35 & -10 of P¬R’
    o Prevents termination of tR
    o Allows RNA Pol to read from PR’ through tR
  4. Extends transcription into late genes downstream of Q
    o Polycistronic RNA with genes for head, tail, lysis proteins needed for lytic cycle
42
Q

What are the requirements for Lysogeny?

A
  1. Monitoring of bacterial health
  2. Repression of Lytic functions
  3. Lambda phage integration
  4. Maintenance of lysogeny
43
Q

Effect of Gene Dosage on Lambda phage lifecycle

A
  • Besides health of bacteria (protease/RNase III activity), CII also depends on gene dosage – how many copies of lambda phage are in the bacterium?
  • If MOI for lambda is 1 phage per cell = insufficient [CII] for activating lysogeny (lytic cycle favored)
  • If MOI is 10 = [CII + CIII] increase = 40-50 fold increase in half life of CII = lysogeny favored
  • Therefore lysogeny depends on health of bacteria + gene dosage of CII and CIII
44
Q

What are the 4 stages of Late Lysogenic Transcription?

A
  1. Monitoring of bacterial health
  2. Repression of Lytic functions
  3. Lambda phage integration
  4. Maintenance of lysogeny
45
Q

Late Lysogenic Transcription - Monitoring of Bacterial health

A
  • In this phase the choice is made for lysogeny by CII
  • CII level related to activity of FtsH/RNAse III and/or gene dosage:
    • Poor growth of bacteria = less bacterial FtsH protease /less RNAse III activity= less CII
    degradation
    • High MOI = high gene dosage of CII/CIII
  • CII protected by CIII
    • Half-life of CII alone = < 1 min
    • Half-life of CII+CIII = ~5 min
  • If CII prevails and protected by CIII, transcription promoted from:
    • PRE
    • PAQ
    • PI
46
Q

Late Lysogenic Transcription - Repression of Lytic Functions

A
  • CII binds to PRE (repressor establishment):
    • CI increases, outcompeting Cro
    • CI binds to OR1 and inhibits transcription from PR = no Cro
    • CI simultaneously binds to PL (O1) = No N protein = no immediate early transcription
    • CI mRNA from PRE (NOT from PRM) is antisense to Cro mRNA
  • CII binds to PAQ
    • 60 bp antisense Q RNA hybridizes to Q mRNA produced from PR
    • dsRNA structure sequester RBS so that the ribosome cannot recognize it = no translation
    • Q protein not translated – late lytic genes not translated
47
Q

Late Lysogenic Transcription - Phage Integration

A
  • CII binds to PI
  • Transcription starts within Xis and terminates at ti = only Int protein produced, no Xis produced
  • Int protein allows integration of phage = prophage
  • Process requires:
    • Integrase and bacterial IHF (integration Host Factor) protein
    • Phage site: attP (POP)
    • Bacterial site: attB (BOB)
    o Located between galactose & biotin operons
  • Site-specific recombination between attP and attB sites via Int and IHF
  • Produces BOP’ and POB’ sequences with prophage in between
  • Difference in order of prophage genes
48
Q

Late Lysogenic Transcription - Maintenance of Lysogeny

A
  • Lysogeny is solely maintained by CI and DNA looping
  • CI binds to OR1 and OR2 and activates transcription from PRM – CI stims own transcription
  • CI continues to repress transcription from PR and PL and leads to decrease in CII and CIII respectively = no activation of PRE, PAQ and PI
  • When CI increases it binds OR3 and inhibits transcription from PRM = neg feedback loop = no CI
  • Normal protein turnover causes CI levels to drop:
    o CI will dissociated from OR3 first – CI bound to OR1 and OR2 will activate transcription from PRM = CI levels increase = maintenance of lysogeny
  • DNA loop is formed by octamerization of CI bound to the OL and OR operator regions and helps maintain a stable lysogenic state in the face of noisy gene expression, non-specific CI DNA binding and operator site mutations
  • Lysogenic Conversion
  • POOP
49
Q

What is the Lysogenic Conversion?

A
  • Part of maintaining Lysogeny
  • Prophage replicates as part of the bacterial genome and its presence changes the phenotype of the lysogen
  • CI not only maintains lysogeny but causes immunity to superinfection by other lambda phages – CI will inhibit their PR and PL promoters
  • 5 other genes also expressed by lambda prophage:
    • Rex A, Rex B, SirB block infection by other phages
    • Lom increases pathogenicity of the lysogen by helping the lysogen bind mammalian cells
    • Bor makes the lysogen more resistant to killing by serum
50
Q

Role of POOP in Maintaining Lysogeny

A
  • This promoter lies in the 3’ end of the CII gene, in the antisense direction
  • the OOP antisense RNA binds to the CII transcript and stims the degradation of the CII mRNA by RNase III
  • It is repressed by the bacterial host LexA protein
51
Q

What is Induction?

A

The transition of a Lambda lysogen from the lysogenic cycle to the lytic cycle

Allows the phage to replicate and leave the host in the face of DNA damage

52
Q

Process of Induction in Lambda-free bacteria

A
  • High stress environment = DNA damage = SOS response
  • SOS response in Lambda-free Bacteria:
    o Damaged DNA activates RecA protease activity (->RecA)
    o RecA     then binds LexA = cleavage of LexA repressor = Damage repair proteins
53
Q

Process of Induction in Lambda lysogen

A
  • High stress environment = DNA damage = SOS response
  • Damaged DNA activates RecA protease activity (->RecA*)
  • RecA* binds CI = cleavage of CI = no dimerization of DNA binding affinity (No looping – was bound to and repressing PL & PR) = PL & PR not repressed = lysogeny removed = excision and lytic growth
  • N protein expressed from PL, allows anti-termination = expression of Int & Xis
  • Cro protein expressed from PR and binds to OR3 = no CI made
  • LexA cleavage allows OOP antisense RNA (antisense to CII) to be made from POOP = binds to CII mRNA and stims degradation by RNase III = no CII protein = no activation of PRE = no CI repressor
54
Q

Lambda prophage excision

A
  • Lambda prophage needs excision for DNA replication to occur
  • Sib site no longer at end of PL transcript in prophage = no hairpin loop = no retroregulation of int and xis
  • Expression from PL allows intact transcript which produces equal concentrations of Int and Xis proteins
  • Both Int and Xis and also IHF required for excision
  • The O, P proteins made from the PR replicate the excised phage Q protein allows expression of the late genes
55
Q

Replication, Packaging, Release from the cell

A
  1. Theta Replication:
    o Initial replication upon entry is theta replication
  2. Rolling circle Replication and Concatemer formation:
    o If lytic pathway or induction from lysogeny occurs then lambda switches to rolling circle replication in latter stages of infection
  3. DNA packaging and Capsid formation:
    o Phage encoded proteins form phage heads
    o Phage encoded terminase binds to cos site and page head
    o Terminase cuts cos sites (leaving 12 bp overhang) and inserts that end into phage head
    o Phage proteins (connector) added afterwards at base of phage head for tail to bind
  4. Tail construction:
    o Tails constructed from 12 gene products and mature tails spontaneously join to the DNA filled capsid
  5. Release from cell:
    o S protein forms a hole in the inner membrane and allows R protein (endolysin) to degrade peptidoglycan cell wall allowing intact lambda phage to be released
56
Q

Rolling Circle Replication and Concatemer Formation

A
  • If lytic pathway or induction from lysogeny occurs tehn lambda switches to rolling circle replication in latter stages of infection
  • Endonuclease cuts 1 strand of the phage DNA – 5’ end of cut plus strand peeled away from intact minus strand
  • Intact minus strand acts as template for DNA replication (dNTPs added at free 3’-OH end of plus strand)
  • Rolling circle replication produces long DNA molecules called concatemers: contain multiple phage genomes

MCB3026F (6 decks)

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