genet 270 lec 16- lambda phage 1
What are the 2 life cycles available to temperate phage like λ?
Lytic (phage replicates, lyses cell) and lysogenic (phage integrates into host chromosome as a prophage).
What are the 2 features distinguishing virulent vs temperate phage?
Virulent phage only perform lytic growth; temperate phage can choose lytic OR lysogenic pathways.
What are the 3 structural features of the λ genome upon infection?
It is linear dsDNA, has 12-bp complementary 5′ overhangs, and circularizes via host ligase sealing the cohesive ends.
What are 3 reasons λ genome organization is functionally important?
Circularization makes the late gene cluster continuous, organizes genes by function, and positions proteins near the DNA sites they regulate
What are the 6 key time points in λ lytic development after infection?
t=0 DNA injection → t=3 early mRNAs → t=5 delayed early mRNAs → t=6 DNA replication → t=9 late mRNAs → t=45 lysis.
(Structural proteins made ~t=10).
What 2 strategies do other phages (not λ) use to regulate host RNAP?
Make their own RNAP (T7) or modify host RNAP to recognize phage promoters (T4). λ instead uses anti-terminators.
What are the 2 early promoters in λ and what do they immediately produce?
PL → N (anti-terminator), PR → Cro (repressor of lysogenic genes).
What are the 3 functions of N protein?
N binds nut sites in mRNA, interacts with RNAP, and enables RNAP to read through intrinsic terminators (tL1, tR1).
What are the 2 major roles of Cro protein?
Cro binds OR/OL to repress cI, and prevents lysogeny by blocking repressor synthesis.
What 3 gene classes are expressed once N allows readthrough of terminators?
DNA replication genes (O, P), recombination genes, and Q anti-terminator.
What are the 3 functions of Q protein?
Q binds qut sites, modifies RNAP, and allows readthrough of tR4 → enabling late gene expression (head, tail, lysis genes).
What were the 2 mutation classes that suppressed N mutations?
Deletions removing tL1, and substitutions replacing DNA containing tR2, so N was no longer required.
What does suppression of N-ambers reveal about λ regulation? (2 insights)
Terminators control timing, and anti-terminators like N allow temporal ordering of early → delayed early gene expression.
What 2 phenomena characterize lysogeny in plaques?
Turbid plaque centers and superinfection immunity (lysogens cannot be infected by more λ).
What 3 reasons explain immunity of lysogens to reinfection?
Lysogens have high CI levels, CI immediately binds OR/OL, and this shuts off PR and PL → no lytic cycle
CI = “See I already live here—no new λ allowed.”
What are the 3 clear plaque mutant classes and their roles?
cI⁻: no repressor → only lytic.
cII⁻: cannot activate cI → fail lysogeny.
cIII⁻: cannot stabilize CII → fail lysogeny.
What 2 categories of vir (virulent) mutations produce clear plaques?
Mutations in OR or OL that prevent CI binding → thus always lytic and can superinfect lysogens.
What are the 2 phases of CI regulation and their promoters?
Establishment via pRE activated by CII, and maintenance via pRM activated by CI itself.
What 3 conditions are required for lysogeny to be established?
Strong CII activity, high initial CI synthesis, and CI concentration exceeding available operator sites.
What are the 2 roles of CIII protein in lysogeny?
CIII protects CII from degradation and stabilizes it long enough to activate pRE.
What are the 2 regulatory modes CI uses to control pRM?
At low CI, it activates pRM; at high CI, it represses pRM via binding OR3.
What are the 3 operator sites and their binding hierarchy for CI vs Cro?
CI binds OR1 > OR2 > OR3 (outside-in).
Cro binds OR3 > OR2 > OR1 (inside-out).
CI binds closest to lytic promoters first to shut them down.
Cro binds closest to pRM first to block CI expression.
What are the 2 consequences of high CI concentration?
Full repression of PR & PL plus repression of pRM, preventing CI overaccumulation.
What 2 outcomes depend on CI concentration?
High CI → lysogeny; low CI → lytic growth.
What are the 3 competing proteins determining CI production early in infection?
Cro, CII, and CIII—Cro represses CI; CII activates CI; CIII stabilizes CII
What is the single most important determinant of lysogeny vs lysis?
CII stability, because CII initiates CI expression from pRE.
(Many slides highlight this as the deciding factor.)
A λ phage lacks CII. What pathway occurs and why?
It undergoes lytic growth, because no CII means no CI expression from pRE → CI never reaches level needed for lysogeny.
CIII is lost. What happens to CII and the lifecycle decision?
CII becomes unstable, decreasing CI production → lytic pathway favored.
Cro is overexpressed early. What happens at OR?
Cro binds OR3 first, blocking pRM, preventing CI production → lytic cycle.
CI is mutated so it cannot bind OR3. What happens to pRM?
pRM becomes hyperactive, producing excess CI → lysogeny persists and induction becomes difficult.
Mutate OR1 so CI can’t bind. Predict early events.
CI cannot block PR → Cro accumulates, repressing CI synthesis and driving lytic growth.
A vir mutant infects a lysogen. What happens?
It proceeds through lytic growth, because vir mutations prevent CI binding → lysogens become superinfectable.
A λ phage is engineered to remove tR1 & tL1. What happens to lytic gene timing?
N becomes unnecessary; early transcription reads through → disrupted timing and premature expression of replication genes.
Cro levels drop unexpectedly early. What happens?
Reduced Cro → less repression of pRM → CI rises → lysogeny more likely.
A mutant RNAP cannot interact with N protein. What fails?
RNAP cannot anti-terminate at nut sites, so delayed early genes (O, P, Q) fail to be expressed → lytic cycle collapses.
A CII-hyperstable mutant appears. Predict phenotype.
Very strong CI accumulation → phage shows high lysogenization frequency and rarely enters lytic cycle.
A phage with defective Cro but normal CI infects the host. What happens?
CI rises unopposed → lysogeny almost always occurs.
A deletion removes OR3. What effect on CI expression?
OR3 cannot bind CI or Cro → pRM is always active → CI always expressed → constitutive lysogeny.
A lambda phage infects a cell already expressing high CI (lysogen). What happens?
Incoming phage is immediately repressed at PR/PL → immunity → no lysis.
Environmental stress destabilizes CI. Predict outcome.
Loss of CI repression → PR/PL turn ON → induction of lytic cycle.
A mutation eliminates qut sites. What happens to late genes?
Q cannot bind → RNAP cannot anti-terminate → no head/tail gene expression → no virion assembly.