Lambda Flashcards
How would you classify a T4 bacteriophage?
What is limiting?
How would you classify a filamentous phage?
Why is it less limiting?
Icosahedral head with tail
Size of head - limited NA
Nucleic acid wrapped in protein (no capsid head)
NA can grow to bigger extent - larger capacity
Lytic Cycle
How does the phage infect the bacterium?
How is it different for filamentous phages?
What occurs in early development?
Late development?
Lysis? With both T4 & filamentous
Interacts with docking proteins with tail proteins & ejects DNA into cell
Uncoat protein & transform nucleic acid - more susceptible to proteases
Genes expressed by RNA polymerase to produce enzymes for DNA synthesis
Genomes replicated, protein head & tail made & DNA packaged into prophages
T4: break cells open & progeny released
F: protein coat added to NA from cell membrane on release
What is lysogenic growth?
How can this be more favourable?
What is lytic growth in comparison?
When is lysogeny favoured?
When is lysis favoured?
How is lysis or lysogeny observed in plaque appearance?
Phage genome integrated into bacterial chromosome
Stable replication in a change of conditions
Aggressive state with release of phage progeny
- Low level of nutrients - still present in bacterial dormancy
- High multiplicity of infection - higher number of phages:bacteria
Making progeny in smallest time frame possible
Lysis = clear plaques as kill bacteria on release of progeny
Lysogeny = turbid plaques (grow in presence of phages over holes)
In what state is the T4 bacteriophage genome?
What do the 12bp cohesive ends do at the end of the DNA?
When is the DNA linear & circular?
What is the largest cluster of genes in the genome responsible for?
How can you describe lytic or lysogenic development?
Linear, double stranded DNA
Overhangs for integration into circular bacteria chromosome (to go from linear -> circular)
Linear in phage. Circular in bacteria
Late genes to form progeny
Controlled at one point via a cascade - like an extreme operon
Lysis Cycle:
Early phase: what promoters does RNA polymerase bind to and what genes are expressed?
Following translation of these into the delayed-early phase, what happens?
What delayed-early genes are transcribed?
What is transcribed in the late phase?
Why does Rolling Circle DNA replication occur?
PL : N
PR : cro
protein N (anti-terminator) binds to nut sites on N and cro sequences to allow host RNA polymerase to access & transcribe delayed-early genes
Q binds to qut sites to allow second anti-termination to transcribe late genes downstream of PR’
Genes to produce head & tail proteins
Go from circular DNA to linear
Lytic cycle continued:
In 4 steps, how does rolling circle DNA replication linearise the DNA on synthesis?
By what system is DNA packaged into the heads & what is it made up of?
In 4 steps, how does the DNA package into an empty head?
- Nick at origin allowing strand to grow from 3’
- Growing from 3’ displaces the 5’ end of the same strand
- Continued revolution of DNA linearises it to its unit length into concatemers which are cut on packaging into heads
- Displaced DNA is replenished & made double stranded by formation of Okazaki fragments
Terminase - proteins NU1 & A
- Terminase recognises 2x cos sites on DNA & binds to the first one
- Translocase system moves DNA into empty head which becomes icosahedral
- Terminase binds to 2nd cos site & DNA cleaves making 12bp overhang
- Tail fibres interact with neck of head & 12bp overhang sticking out of head to form final progeny
What phase do lysogeny & lysis cycle both share?
What does cro do?
What does N anti-terminator allow? 2 things
When N binds to nut sites, what genes are transcribed in delayed-early phase?
What does Q allow?
Immediate early - making of N & cro proteins
Prevents expression of c1 (lambda repressor)
Expression of c2-c3 genes needed to synthesise c1, and allows expression of Q (anti-terminator of late genes in lytic)
C2 & C3
Binds to qut sites which overlaps with PR’ - allowing for host RNA polymerase to transcribe late genes
How is the lambda repressor (c1) expressed?
Why?
What does c3 do?
What does the lambda repressor bind to once synthesised? 2 things
What are the two promotors after the transcription of delayed early genes (c2/c3) responsible for?
N binds to nutR - expressing C2 - binds to the PRE promotor leading to lambda establishment (expression c1)
PRE is a weak promoter so requires C2 regulator
Protects degradation of c2 (for transcription of c1)
- Binds to OL/OR to inhibit transcription of early genes
- Binding to OR activates PRM for repressor maintenance/auto-regulation
PRE - establishing c1 expression
PRM - maintaining c1 expression
How does the lambda repressor bind to operator sites?
What else does the lambda repressor inhibit?
What is the C-terminal domain responsible for?
N-terminal domain?
How does dimerisation increase the repressor’s affinity for DNA?
The 14-residue linker between the domains is susceptible to what?
What could cleavage of this do?
As a dimer
Transcription of DNA of incoming phages on infection as well as its own early genes
Dimerisation & association with another monomer’s C-terminal domain
DNA-binding domain with high affinity
Orientation/conformation caused by the C-terminal interactions
Proteases, UV, changing conditions
N-terminal domains not remain in dimerised form, so weaker affinity to DNA, repressor released from operators so inducible for lytic cycle to continue & lysogeny to end
What is the entry to lysogeny?
What protease can degrade C2 & what does degradation result in?
What would a mutation in the following cause:
c1
c2
c3
Expression of c2 (& c3) and formation of c1 & preventing transcription of early genes so inhibiting cascade into late genes
High frequency lysogeny protease - c2 degradation means won’t bind to PRE & c1 not established so higher likelihood of lytic cycle
c1: inactive lambda repressor = lysogeny
c2: difficult establish lambda - can’t bind to PRE
c3: can’t protect c2 so degraded - lower levels of c1
From which promoters include cro in their mRNA transcript?
How is translation of cro inhibited?
Why is the production of this structure favoured in lysogeny?
C2 is a positive transcriptional regulator of 3 promoters. For each promoter, describe what they are used for:
PRE
PI
PantiQ
PR (+ c2) sense and PRE (+ c1) antisense
Through antisense hybridisation - hydrogen bonds between complimentary overlap of cro mRNA to form double-stranded mRNA region
Cro inhibits c1 - so preventing its translation means C2 can form C1 for its own maintenance
PRE: initiate c1 expression & antisense cro (which forms antisense hybridisation with PR transcript)
PI: increase expression level of integrase for integration of phage DNA into host chromosome
PantiQ: Makes antisense transcript with Q mRNA (and mRNA containing excisionase) to prevent transcription of late genes/excision from chromosome
By what method is the phage DNA integrated into the host chromosome?
What is required?
What structure is required for integration to occur?
What are the 5 steps for chromosome integration?
What else is required for excision (lysogeny -> lysis)?
In what condition is this enzyme made?
Site specific recombination
Complimentary sites attB (bacteria) and attP (phage), high concentrations of integrase (PI) and IHF (integration host factor)
Intersome
- Host IHF recognises intersome structure & wraps around surface to expose the complimentary region
- integrase interacts with attP & IHF forming an active intersome
- phage DNA is therefore exposed on the active intersome surface for interaction with integrase
- allows recruitment of bacterial chromosome for recombination
- 1 strand of bacterial DNA cut at a time & re-joined to the other end of the phage DNA
Excisionase
When no antiQ is present - so Xis gene isn’t within hybridised mRNA transcript & can be translated
Which sites does the lambda repressor have highest affinity for?
What kind of binding is exhibited?
What does binding of lambda to O1 and O2 do?
What does lambda binding to OR2 achieve?
What about lambda binding to OR3?
Why is this dangerous for lysogeny?
OR1 & OL1 (2x half sites per operator)
Cooperative binding - easier to bind to OR2 & OL2
Inhibits expression of PR (cro) & PL (N)
Allows Host RNA polymerase to bind to PRM - increases affinity for it for own self-maintenance of c1
Obscures RNA polymerase - inhibiting transcription of PRM until concentration of lambda falls - to which lambda will fall off OR3 to re-regulate PRM
Turning off auto-regulation can increase chance going back to lytic state
What are the properties of cro?
How is it different to lambda?
How is this dangerous for lysogeny?
What can cro do after binding to OR3 & OL3?
What has already happened before cro can do this?
In what stage does Q accumulate?
Dimer (like lambda), but smaller, helix-turn-helix binding domain allows interaction with DNA/operators
Exhibits no co-operative binding to binds to OR3 first with high affinity due to helix-turn-helix binding domain
Binding to OR3 inhibits transcription at PRM so turns off autoregulation of c1 lambda repressor
Can then bind to O2 & O1 sites - turning off PL & PR expression
Protein Q has been made (from early genes) but there is not sufficient amount of C2 to form anti-Q transcript: allows for transcription of late genes from PR’ promoter
Delayed early - through N binding to nut sites to anti-terminate transcription of the genes (c2, c3, Q)
What is the main balancing force between lysis and lysogeny?
How does a high concentration of c2 induce lysogeny?
How does a high cocentration of cro induce lysis?
What can impact c2 concentration in a cell? (2)
Concentration of C2 & concentration of cro
Turn on PRE (C1), turns down cro expression with antisense hybridisation complex, induces P anti Q preventing translation late genes, induces PI for lysogeny
Binds to OR3 preventing PRM self maintenance, then O2/O1 inhibiting PL/PR of early genes, then Q anti terminator allows expression late genes & excision
- Cell dormancy with fewer proteases to degrade C2
- Location of phage infection - at poles have high concentration of proteases
