Bacteriophage lambda Flashcards
What is bacteriophage lambda?
A virus of bacteria
phage = nucleic acid + coat (capsid)
What are common shapes of phages?
Icosahedral
Filamentous
Head and tail
How do virus’ attack host cells?
They inject their genetic material into the host cell
What are early genes for?
Things that need to be done first
Encode proteins involved in switching on late genes
What are late genes for
Things that need to be done last
What are the two main cycles that phage lambda could follow?
Lysogeny cycle - DNA integrates into host chromosome
Lytic cycle - new phage particles are made. The host is killed to allow these to burst out.
What controls which cycle bacteriophage lambda enters?
How many phage there are per bacterial cell
How much food is available
How healthy the bacteria are
What is an induction event?
External stress (UV damage) causes the phage to try and exit the E.coli cell. Incorporation of the phage DNA into the host is reversed. /9
What happens in the pro-phage pathway?
The phage DNA is incorporated into the bacterial host’s DNA to create the prophage. This requires the enzyme integrase which is encoded by early genes
Stage 1 of the pro-phase pathway
The circular chromosome of bacteriophage lambda has an attachment site sequence. The bacterial chromosome has a matching attachment sequence. There is a particular site for the viral DNA to incorporate.
Stage 2 of the pro-phase pathway
Integrase binds the bactreiophage at the attachment site then binds the matching attachment site of the bacterial DNA
Stage 3 of the pro-phase pathway
DsDNA breaks in phage and E.coli
Asymmetric cut creates sticky ends
Cut sites are re-joined and they are slightly over-lapping by 7 nucleotides
What is important about the lytic pathway
That the timing of events are right to get the bacterium to make all the regulatory proteins needed for the control of the expression.
In the lytic pathway how is transcription regulated
Positive and negative regulation
What are the name of the promoters in the lytic pathway?
PL & PR
What is the order of genes involved in lytic pathway?
Int xis CIII tL1 N PL PR tR1 Cro CII PLE O tR2 P Q tr3 S R
In the lytic pathway what are the first genes to be transcribed?
N & Cro
Then
CIII, CII, O, Q if the antiterminator is present
Function of Cro
Makes a regulatory protein
Funciton of N
Encodes an antiterminator which will attach to the nutL & nutR sites which RNA polymerase will pick up as it passes.
Function of Q
Encodes an antiterminator protein
What genes are expressed after anti-termination?
O,P: Replication of phage genome
Q: encodes antiterminator
S,R: late genes, head & tail, lytic enzymes
How is lysogeny established?
CI encodes the lambda repressor.
CIII & CII are positive control factors that enable the production of the lambda repressor
What does the lambda repressor do?
Inhibits the ecpression of the left and right operons by binding to the two promoter regions to prevent transcription.
PRM is the promoter for repressor maitenance. Lambda repressor acts on PRM to cause transcription of CI which produced more lambda.
Explain the role of degradation on CII
Proteases encoded by bacterial host genes (HflB- high frequency of lysogeny) have an inhibitory effect on CII to cause degradation. CIII will deactivate HflB. CII can continue to activate the promoter for repressor establishment.
What happens with degradation if the bacterial host cell is growing quickly?
CIII is repressed = decline in CII = decline in lambda = lytic cycle
Function of O,P
Replication of the phage genome/ initiate phage replication
Function of S,R
Late genes, head/tail, lytic enzymes e.g. endolysin which digests bacterial cell walls
Cro V lambda repressor
Cro aims to promote the lytic cycle and prevents the lambda repressor from binding to PR or PL operator regions
lambda repressor aims to promote lysogeny
What are Cro and lambda competing for ?
Operator region
What does Cro stand for?
Curtailment of repressor and other things
How does lambda bind to the operator regions of the right promoter?
OR1 coincides with the promoter region for the rightward operon. Lambda preferentially binds to OR1 and once bound it represses this promoter region to repress the lytic genes.
Lambda repressor then binds OR2 to stimulate RNA polymerase to start transcribing from PRM which maintains lambda function by promoting its own production.
Lastly, lambda will bind OR3 when concentration is especially high which blocks its own promoter to switch off its production.
How does Cro protein bind to the operator regions?
Cro will preferentially bind to OR3 which blocks the lambda promoter to shut down lysogeny.
Binding to OR2 & OR1 follows which then shuts down its own promoter region
How do high levels of CII promote lysogeny?
Activate PRE
Prevent the late gene anti-terminator Q from forming
Can also make an antisense transcript which cancels out the normal Q transcript so there is no antitermination
What is meant by “the switch”?
The switch between lysogeny and the lytic cycle
How was the switch discovered?
Phage plaques: clear area on lawn of bacteria where the phage has gone in a lysed the bacteria.
Mutants of phage lambda were going straight into the lytic cycle and there was no lysogeny. The mutants were identified to be in one of the genes: CI, CII, CIII
What is a lawn of bacteria?
When there are so many colonies that they just merge together
What makes a cell switch from the lysogeny pathway to the lytic pathway?
Proteolytic activity of RecA protein splits up the lambda repressor into 2 halves.
Early gene expression in the left and right operon can then occur.
What is the switch part of?
The SOS response
What may cause the switch?
DNA damage results in ssDNA. RecA recognises ssDNA which activates its protease activity. The protease can cleave the lambda repressor to allow activation of SOS genes.
What occurs as lysogeny is broken?
Integrase and excisase remove the prophage.
Replication enzymes and endolysin are produced
