5) Bacteriophage lambda Flashcards
(34 cards)
What are bacteriophages? What is their structure?
These are viruses that infect bacteria
Phage= Nucleic acid + coat (capsid)
-Bacteriophage lambda forms the head-and-tail coat
-Double stranded (ds linear DNA chromosome)
How do viruses reproduce themselves?
- Viruses use their host’s molecular machinery to reproduce themselves
- Different proteins are needed at different stages of the program
- So different genes need to be expressed at different times (early/late)
1) Viral genome enters a host cell
2) It is then replicated to produce multiple copies= Transcribed and translated to produce viral coat protein
3) Viral genomes can assemble spontaneously with the coat protein= Form new virus particles, which escapes from the cell by lysing it
How do bacteriophages co-ordinate the expression of genes to facilitate transition through stages?
1) First genes to be expressed (usually after infection) produce a protein
2) The protein will allow the switching on of genes that are needed later= Later genes
What is the bacteriophage lambda?
Bacteriophage which infects E.coli cells
What are the two different choices which phage lambda can make when it injects its DNA into the E.coli cell?
1) Phage lambda injects its DNA into E.coli cell
2) Its DNA re-circularises
CAN EITHER:
3) Lysogeny= DNA integrates into host chromosome
Lysogeny= Also called prophage, phage’s DNA is replicated for it by the bacterium
OR
3) Lytic= New phage particles made, host is killed to allow these to burst out
Aim: To create many more phage particles to infect other cells, requires synthesis of proteins required to kill the host AND build new phages= Needs specific genes to be switched on
It is a molecular switch
When would it choose lysogeny instead of lytic?
If there are not many unoccupied hosts OR not much nourishment around for more bacteria that the virus could invade= Best to stay in host
Host with lysogen= Immune to further infection= New phages can be adsorbed to its surface of an already infected bacterium but their DNA will not be taken up in the host chromosome
When would it choose lytic instead of lysogeny?
If the bacteriophage is going to make lots of new phage particles, it is only sensible to do this if there are lots of new bacterial hosts for them to invade
Which option do virulent phages always go straight to?
They are active phages and they always go for lytic option whereas temperate phages can do either
When do early genes need to be transcribed? What do they do?
As soon as the lambda DNA plasmid has recircularised
Genes direct the synthesis of viral proteins needed for the formation of new viruses such as coat proteins
When do late genes need to be expressed?
Need to be expressed later before the assembly of more virus particles and their release from the bacterial cell by lysis
What happens during the lysogeny (prophage) pathway?
Example of conservative site-specific recombination
1) 2 homologous DNA sequences (attachment sites) are present in the phage and bacterial DNA
2) Phage produces integrate enzyme which binds to specific phage sequences on phage chromosome
3) Integrase then makes 2 cuts in both chromosomes on either side of the region
4) Integrase then switches the partner strands, reseals them
5) Forms a small heteroduplex joint that is 7 nucleotides long (as 7 basses of DNA from the phage is matched with 7 from the host)= Phage DNA is integrated into the bacterial host’s DNA
How can the integrase enzyme know when it needs to act or not?
It can recognise these new joins as being different from the original sequence in the 2 separate plasmids= can detect whether it needs to act or not
What enzymes helps integrase if the DNA needs to be removed from the bacterial chromosome?
Excisase
What happens during the lytic cycle?
1) Late genes must be transcribed for this to occur
2) Synthesis of viral proteins needed for formation of new viruses
3) Rapid replication of lambda DNA at its packaging into complete viruses
4) Cell lysis releases a large number of new viruses
What types of regulation of transcription does it use?
Both positive and negative
An absence of activation is NOT the same as switching off completely= Nothing is absolute!
Balance between the two options= Determines the outcome + nothing happens instantly
Mostly negative regulation or relief of that inhibition= Only 2 examples of positive regulation
What are the 3 proteins which are involved in transcription of the phage? What happens when they are mutated?
CI, CII, CIII
Mutated= Phage will always go to lytic cycle
Normal: Each of these 3 proteins are either positive factors directing phage towards lysogeny or factors that repress the lytic cycle= Normal= Most of time is in lysogeny
How is transcription stated for bacteriophage lambda?
1) Immediately upon infection and circularisation of phage genome= Bacterial RNA polymerase attaches to 2 promoters PL and PR
2) RNA polymerase start transcribing the leftwards operon and rightwards operon, ending at transcription termination signals tL and tR1
3) Early transcription: 2 genes are transcribed, N and CRO
4) N is ANTITERMINATION TERMINATOR by interacting with RNA polymerase at Pl and PR1 to allow it to pass through the initial terminators and transcribed left and right operons
What does the action of the protein ‘N’ antiterminator allow RNA polymerase to do?
Allows one terminator signal to be ignored on the leftwards operon and 2 on the rightwards operon= More operons can be transcribed
What is the different between the leftwards and rightward operons?
Genes are arranged as a huge operon around the circular plasmid = Phage ‘decides’ between the 2 options of lytic and lysogenic
Leftward operons:
MAINLY to do with lysogeny
Rightward operons:
Lytic pathway
What does the lambda (λ) repressor do?
It represses the lytic cycle and allows the establishment of lysogeny
It is cI gene
NOTE: Not under control of either the leftwards or rightwards promoter= Has its own promoter somewhere else in order to function
How is cII stabilised?
Stabilised by cIII protein
•cIII acts as an inhibitor of the bacterial host’s own proteases that destabilise cII
•There are 2 proteases: HflA and HflB that both destabilise cII (Hfl= High frequency of lysogeny)
•If these proteases are inactivates= cII is more stable
•Expression of HflA and HflB proteins is subject to catabolite repression
•Sugar starvation (energy deprivation)= Leads to higher frequency of lysogeny due to less activity of the Hfl proteases
•Tendency to estabilish lysogeny is dictated by the growth conditions of the bacteria
•cII= Stimulates transcription from the PRE promoter= Repressor establishment= Allows the expression of the cI gene which encodes the lambda repressor
What does the λ repressor do to leftward and rightward operons?
Inhibit the expression of them= Including cII and cIII
Useful: Switching cI expression in the first place
How are cI levels kept high enough to maintain lysogeny?
λ repressor: Blocks pL and pR= cIII and cII are not expressed as their operons are no longer expressed
Lambda repressor: POSITIVELY regulates its own transcription
Able to act as a positive control factor + Stimulate own expression from PRM promoter= Promoter for repressor maintenance
What happens when there is a continued expression of the λ repressor protein?
Makes: Lysogen= Bacterial strain carrying a prophage, immune to further infection by the same type of phage
Good for phage: Doesn’t have to compete with other phages
Good for bacteria: Immune to further infection
