Phage Lambda Flashcards
What is phage lambda?
a temperate bacteriophage that infects E. coli
What is lytic infection?
the process by which a phage infects a bacterium, reproduces itself and then kills its host
What happens in a typical lytic cycle?
- phage DNA enters host bacterium
- its genes are transcribed in a set order
- the phage genetic material is replicated
- the protein components of the phage particle are produced
- host bacterium is lysed to release the assembled progeny particles via lysis
What is lysogeny?
the ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome
What is prophage?
a phage genome covalently integrated as a linear part of the bacterial chromosome
What is phage induction?
when the prophage switches from the lysogenic to the lytic cycle
What are the 2 phage types?
- temperate - can choose between a lytic and lysogenic pathway
- virulent - lytic and unable to display lysogenic changes
What are the 2 periods of a phage infective cycle?
- early infection - period from entry into DNA to start of replication
- late infection - period from start of replication to final step of lysing
How does the phage undergo protein synthesis?
it uses the host apparatus i.e. redirects its activities by replacing bacterial mRNA with phage mRNA
What may regulator proteins used in phage cascades do?
- sponsor initiation at new promoters
- cause the host polymerase to read through transcription terminators
What does control at initiation of replication use?
independent transcription units, each with its own promoter and terminator, which produce independent mRNAs
What does control at termination of replication require?
adjacent units so that transcription can read from the first gene into the next gene
What 2 possible forms can the regulator at each stage of phage expression take?
- new sigma factor that redirects the specificity of the host RNAP
- antitermination factor that allows new groups of genes to be read
What is the early phase before replication split into?
immediate and delayed stages
What are the 2 lambda immediate early genes?
N and cro transcribed by host RNAP
What does N gene do?
express the delayed early genes
What are the 3 regulatory delayed early genes?
- cII
- cIII
- Q
What genes do lysogeny and the lytic cycle require respectively?
- lysogeny = cII and cIII
- lytic cycle = cro and Q
What is the regulator of antitermination?
pN protein (encoded by N immediate early gene)
What does pN protein do?
allow transcription to continue into the delayed early genes by suppressing use of the terminators tL and tR
What happens in the presence of pN?
transcription continues to the left of the N gene into the recombination genes and to the right of the cro gene into the replication genes
What does lambda DNA do during infection?
circularise so that the late gene cluster is intact in one transcription unit starting from a promoter PR’ between Q ands S
Describe the late gene cluster
lysis genes S-R are at the right of the linear DNA and the head and tail genes A-J are at the left
What does gene Q allow?
RNAP to initiate at PR’ to transcribe the late genes (antitermination factor)
What happens in the absence of gene Q?
late transcription terminates at a site tR3
Describe what happens in early, delayed early and late stages of phage lambda infection
- early - host RNAP transcribes N and cro from promoters PL and PR
- delayed early - pN allows transcription from the same promoters to continue past N and cro
- late - transcription initiates at PR and pQ allows it to continue through all the late genes
What encodes the lambda repressor?
delayed early gene cI
What is the lambda repressor required for?
to maintain lysogeny
What are the 2 promoters of the cI gene?
- PRM (promoter right maintenance)
- PRE (promoter right establishment)
Where does the lambda repressor act?
at the OL and OR operators to block transcription of the immediate early genes
What happens when immediate early genes are repressed?
the lytic cycle cannot proceed
What does phage lambda use antitermination for?
to proceed to the next stage of delayed early expression
Where do the promoters PL and PR lie?
on either side of the cI gene
What does the lambda repressor do?
- prevent RNAP from initiating transcription at PL which stops expression of N
- prevents use of PR at OR1 i.e. cro and other rightward early genes can’t be expressed
What does lambda repressor binding at OR1 stimulate?
transcription of cI
What are the 2 domains of a repressor monomer?
- N-termial domain with DNA binding site
- C-terminal domain that dimerises
What does the lambda repressor binding to the operator require?
the dimeric form of the repressor so that 2 DNA-binding domains can contact simultaneously
What does cleavage of the repressor between the 2 N-terminal domains do?
- reduce the affinity for the operator
- induce a lytic cycle
What do virulent mutations do?
prevent the repressor from binding at OL and OR so that it is unable to establish lysogeny
What is the serial dilution method for counting phages?
- as the phages grow and lyse the host, holes/plaques are formed in the bacterial lawn
- the plaques can be counted and the original concentration of phage can be determined
Why are plaques cloudy with wild type phages?
they contain some cells that have established lysogeny instead of being lysed
Why are plaques clear with virulent mutants?
they are unable to establish lysogeny i.e. plaques contain only lysed cells
What does repressor binding to one operator do?
increase the affinity for binding a second repressor dimer to the adjacent operator
Why are the OL1 and OR1 operators bound first?
cI has 10x greater affinity
What does repressor binding at OL and OR do respectively?
- OL - blocks transcription of gene N from PL
- OR - blocks transcription of cro but promotes transcription of cI
What is the basis for the autoregulatory control of repressor maintenance?
the DNA-binding region of repressor OR2 contacts RNAP and stabilises its binding to PRM
In the lysogenic state, what do the repressors bound at different operators interact with?
- OL1 and OL2 interact with those bound at OR1 and OR2
- RNAP is bound at PRM and interacts with the repressor at OR2
What are the delayed early gene products cII and cIII required for?
RNAP to initiate transcription at PRE
What do cII and cIII do respectively?
- cII - acts directly at the promoter
- cIII - protects cII from degradation
What does transcription from PRE lead to?
synthesis of repressor and blockage of cro synthesis
When will RNAP bind to PRE?
only in the presence of cII at the -35 region (cII binding site)
What does the cI gene do at lower and higher concentrations of repressor?
- lower - forms the octamer and activates RNAP in positive autogenous regulation
- higher - binds to OL3 and OR3 and turns off transcription in negative autogenous regulation
In what 2 ways does transcription from PRE promote lysogeny?
- directly by cI mRNA being translated into repressor protein
- indirectly by transcription proceeding through the cro gene in the wrong direction
How much more efficiently is the cI coding region of the PRE transcript translated than the cI coding region of the PRM transcript and why?
7-8x since the PRE transcript has an efficient 5’ UTR containing a strong ribosome-binding site
What 4 events does lysogeny require?
- cII and cIII establishing repressor synthesis and triggering inhibition of late gene transcription
- establishment of repressor turns off immediate and delayed early gene expression
- repressor turns on the maintenance circuit for its own synthesis
- lambda DNA is integrated into the bacterial genome
What is the cro repressor required for?
lytic infection
What does the cro repressor do?
- directly prevents repressor maintenance via PRM
- turns off delayed early gene expression
- prevents synthesis of cI
What does cro repressor bind with the highest and lowest affinities?
- highest - OR3/OL3
- lowest - OR1/OL1
What happens when cro repressor binds to OR3?
it prevents RNAP from binding to PRM
What is the critical event of the delayed early stage?
whether cII causes sufficient synthesis of repressor to overcome the action of Cro repressor
