genet 270- lec 17- lamda phage 2
What are the 3 major features of λ lytic growth that differ from T7/T4?
λ uses host RNAP (does not make its own), modifies termination rather than promoter specificity, and uses anti-terminators N & Q to extend transcripts.
What are the 2 ways λ prevents wasteful early gene synthesis during late gene expression?
Cro represses PL and PR, stopping early transcripts once late genes (pR2/pR′) turn on.
What are the 3 essentials of λ DNA replication (based on slide 3 image)?
Origin oriR in gene O, O/P recruit and “pirate” host DnaB, and replication shifts from θ to rolling circle, producing concatemers. Gam inhibits RecBCD to allow RCR.
Memory tip: O = “Origin organizer,” P = “Pirate of helicase.”
What are the 2 molecular balances that determine lysogeny vs lytic growth?
N vs CII/CI and Cro vs CI competition define whether repression or lytic transcription dominates.
What are the 3 key functions of CII in lysogeny?
CII activates pRE (CI synthesis), pI (integrase), and is stabilized by CIII which protects it from HflA/B proteases.
What are the 2 reasons CII stability is crucial?
CII has a short half-life (~2 min) and is degraded by HflA/HflB, so only stable CII can activate CI and initiate lysogeny.
Connection: CII is the “decision node” because CI expression depends on CII.
What 3 conditions increase probability of lysogeny?
High MOI, host mutants that stabilize CII (e.g., HflA/HflB defects), and starvation before infection (increases lysogeny 50–100×).
According to Table 8.3 (page 6), what are 4 steps leading to lytic growth vs lysogeny?
Lytic: N & Cro expressed → CII degraded → low CI → PR/PL ON → replication.
Lysogeny: CII stabilized → CI expressed → PR/PL OFF → integration occurs.
What are the 3 biochemical activities of Integrase (Int)?
Int binds attP, is a type I topoisomerase, and catalyzes site-specific recombination between attP and attB.
What is an intasome and what 2 components does it require?
A protein–DNA complex containing Int + both att sites (attP & attB) brought together for recombination.
What are the 4 mechanistic steps of attP × attB recombination?
Int nicks both sites, strand exchange occurs, a Holliday junction forms, and resolution produces BOP′ + POB′ (recombinants).
Why is Int synthesis tied to CII activation? (1 reason)
CII activates pI, allowing efficient integrase expression only when conditions favor lysogeny.
Why can Int alone NOT excise λ prophage? (2 reasons)
Excision requires Xis, which alters DNA binding specificity and enables Int to recombine attL & attR instead of attP/attB.
What are the 3 molecular functions of Xis?
It is an excisionase, forms a complex with Int, and promotes recombination at attL/attR (reverse reaction).
Why is Xis NOT produced during lysogeny? (3 explanations)
CI shuts off PL, CII activates pI (inside xis gene → no Xis), and RNA degradation prevents Int/Xis buildup during integration.
What is retroregulation? (Define in 1 sentence)
Regulation in which sequences downstream of a gene control stability of its mRNA, here affecting int/xis RNAs.
What are the 3 steps of retroregulation after initial infection?
PL transcript extends across attP via N; RNase III cleaves; RNase II degrades downstream RNA, eliminating int/xis transcripts.
Why does retroregulation NOT operate in prophage? (1 key explanation)
The genome is reorganized so the RNase III site is no longer adjacent to attB/P, so PL transcripts now produce stable xis RNA after induction.
“Circular hides the cut site” — once integrated, RNase III can’t clip Int/Xis RNA until induction.
What are the 3 events triggered by DNA damage that initiate λ induction?
ssDNA activates RecA*, CI undergoes autocleavage, and PR/PL turn ON, expressing N, Cro, Int, and Xis.
Why are RecA⁻ lysogens non-inducible? (1 reason)
RecA* is required for CI cleavage, so without it λ cannot exit lysogeny.
During induction, why are Int and Xis expressed stably? (2 reasons)
PL is ON and the RNase III cleavage site is repositioned, so PL transcripts are not degraded.
What are the 3 environmental or intracellular factors that influence the decision?
Nutrient state, host protease levels (HflA/B), and multiplicity of infection.
What does Figure 7.24 illustrate about CI vs Cro? (2 concepts)
High CI → lysogeny (PR/PL OFF), high Cro → lysis (CI OFF). Each represses the promoter of the other.
What are the 3 sequential stages illustrated in the decision diagram?
Early infection (N, Cro expressed), competition phase (CII stability determines CI), and final outcome: lytic (Cro wins) or lysogenic (CI wins).
CII is the decider, CI is the maintainer, Cro is the lytic promoter.
What is the 1 experimental way to distinguish vir mutations from cI, cII, cIII mutations?
Test ability to infect lysogens: vir mutants form plaques on lysogens, while cI/cII/cIII mutants do not.
Why can λ vir (OR1/OL1) mutants form plaques on λ lysogens? (2 reasons)
Vir mutations prevent CI binding, so incoming phage ignores immunity and undergoes lytic growth.
Why are some prophages inducible only by infection with another phage? (1 concept)
Superinfection provides necessary anti-repressors or additional regulatory factors to overcome strong immunity and trigger induction.
HflB protease is overexpressed. What happens to λ development?
CII is rapidly degraded → CI is not made → lytic cycle favored.
CIII is deleted. How does this affect lysogeny frequency?
CIII no longer protects CII → CII unstable → lysogeny drops dramatically.
Mutate pI promoter so Int cannot be expressed. What outcome?
λ cannot integrate → only lytic cycle occurs, even if CI is produced.
A strain lacks RNase III. What happens during initial infection?
PL transcript not degraded → Int/Xis accumulate prematurely → integration becomes inefficient.
CI is mutated to resist RecA cleavage. What phenotype results?
CI cannot be inactivated → prophage becomes non-inducible (defective induction).
Xis is overexpressed during early infection. Predict effect on lysogeny.
Xis promotes excision → prophage cannot stabilize → lysogeny fails
attB is deleted from the host chromosome. What happens?
λ cannot integrate → strictly lytic infection
CII is mutated to resist degradation. What phenotype results?
Very high CI production → lysogeny frequency skyrockets.
Cro cannot bind OR3. What happens to pRM?
Cro fails to repress pRM → CI continues being produced → lysogeny more likely.
A λ phage has a mutated N protein unable to bind nut sites. Result?
Early termination cannot be bypassed → delayed early genes (O, P, Q) not expressed → lytic cycle collapses.
DNA damage occurs but RecA activation is weak. Predict outcome.
CI cleavage insufficient → partial induction or no induction → prophage remains lysogenic.
After induction, PL transcripts become extremely short due to a new terminator. What effect?
Xis/Int levels drop → defective excision → phage cannot efficiently enter lytic cycle.
High MOI infection in nutrient-poor media. Predict outcome.
CII stabilized + multiple phage genomes → lysogeny strongly favored.
attL or attR is mutated. What fails first: integration or excision?
Excision fails, because attL/attR are excision-specific recombination sites.
A cI⁻ donor infects a λ lysogen. What happens?
Incoming phage lacks immunity target → lysogen’s CI represses it → no productive infection.