5) Bacteriophage lambda

Question 1

What are bacteriophages? What is their structure?

Answer 1

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)

Question 2

How do viruses reproduce themselves?

Answer 2

• 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

Question 3

How do bacteriophages co-ordinate the expression of genes to facilitate transition through stages?

Answer 3

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

Question 4

What is the bacteriophage lambda?

Answer 4

Bacteriophage which infects E.coli cells

Question 5

What are the two different choices which phage lambda can make when it injects its DNA into the E.coli cell?

Answer 5

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

Question 6

When would it choose lysogeny instead of lytic?

Answer 6

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

Question 7

When would it choose lytic instead of lysogeny?

Answer 7

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

Question 8

Which option do virulent phages always go straight to?

Answer 8

They are active phages and they always go for lytic option whereas temperate phages can do either

Question 9

When do early genes need to be transcribed? What do they do?

Answer 9

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

Question 10

When do late genes need to be expressed?

Answer 10

Need to be expressed later before the assembly of more virus particles and their release from the bacterial cell by lysis

Question 11

What happens during the lysogeny (prophage) pathway?

Answer 11

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

Question 12

How can the integrase enzyme know when it needs to act or not?

Answer 12

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

Question 13

What enzymes helps integrase if the DNA needs to be removed from the bacterial chromosome?

Answer 13

Excisase

Question 14

What happens during the lytic cycle?

Answer 14

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

Question 15

What types of regulation of transcription does it use?

Answer 15

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

Question 16

What are the 3 proteins which are involved in transcription of the phage? What happens when they are mutated?

Answer 16

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

Question 17

How is transcription stated for bacteriophage lambda?

Answer 17

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

Question 18

What does the action of the protein ‘N’ antiterminator allow RNA polymerase to do?

Answer 18

Allows one terminator signal to be ignored on the leftwards operon and 2 on the rightwards operon= More operons can be transcribed

Question 19

What is the different between the leftwards and rightward operons?

Answer 19

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

Question 20

What does the lambda (λ) repressor do?

Answer 20

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

Question 21

How is cII stabilised?

Answer 21

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

Question 22

What does the λ repressor do to leftward and rightward operons?

Answer 22

Inhibit the expression of them= Including cII and cIII

Useful: Switching cI expression in the first place

Question 23

How are cI levels kept high enough to maintain lysogeny?

Answer 23

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

Question 24

What happens when there is a continued expression of the λ repressor protein?

Answer 24

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

Question 25

What genes does the rightward operons contain?

Answer 25

Contains the genes:
CRO: Regulatory gene= Inhibits transcription from leftwards operon , High conc= Reduce expression of the rightward operon
cII: Switched on as soon as the first anti terminator signals could be ignored with help of N
CRO and cII= Encode proteins needed for making more phage and killing the cell so phage can escape

O and P= Directing replication of the phage genome

Q= Switches on the major rightwards operon that expresses all of the head and tail protein genes + Anititerminator that allows RNA pol to transcribe even further in rightwards direction, and these genes encode genes for head proteins to make capsid

S and R= Enzyme that lyses the bacteria from the inside

Question 26

What is the difference between CRO and cI (λ) repressor?

Answer 26

λ repressor= Inhibits leftwards AND rightwards but promotes itself, AIM= Cause lysogeny pathway to be adopted

CRO= Blocks binding of λ repressor at the 2 promoters , AIM= Promote lytic pathway

Promoters= Have operator regions, where repressor proteins will bind to have their effect and stop RNA pol transcribing genes

λ and CRO are in conflict!

Question 27

How does λ repressor actually prevent the transcription of the rightwards operon and regulate its own transcription?

Answer 27

1) cI protein binds to OR1 (binding affinity is highest)
2) OR region overlaps with the rightward promoter (PR)
3) Inhibits the transcription of the rightwards operon= All of lytic cycle genes AND CRO

1) Binding to OR1 stimulates binding of cI protein to OR2
2) When OR2 is bound by λ= Helps RNA pol to bind to PRM and helps stimulate expression from PRM
3) High concentrations of cI protein OR3 bound= inhibits further transcription from PRM= Blocked and RNA pol cannot access PRM

Question 28

How does CRO work?

Answer 28

Binds to the same operators as λ, BUT IN REVERSE as binding affinities are reverse

1) First binds to OR3= Prevents expression of cI as it blocks PRM
2) Then binds to OR2= Prevents cI protein from stimulating its own expression as cI cannot get there
3) Then binds to OR1= Reduces expression of rightwards operon

Question 29

What makes a cell switch from lysogeny to the lytic phase?

Answer 29

Nearly always: λ repressor always wins= Need alternative pathway to happen to be removed

BUT: CRO gets there first= Decision is made between early and late expression

Question 30

How does a cell switch from lysogeny to the lytic phase?

Answer 30

INDUCTION EVENT:
Breaking lysogeny= Waking the system up so that it can change from the dormant lysogeny state to the lytic state

NEED: to stop cI inhibiting both directions, can do this by having excisase to release the phage DNA from the lysogen and need the rightward operon to be expressed so we can get all the proteins needed for phage coat proteins, lysis etc…

Question 31

Under what conditions does breaking lysogeny happen?

Answer 31

Rare event: Only happens under conditions of stress

Particularly stress that can damage DNA such as UV light, carcinogens and mutagens= Elicit SOS response

Question 32

What is the SOS response? (just definition)

Answer 32

An army of enzymes that can repair damaged DNA, normally kept repressed by the product of the lexA gene

RecA is expressed

Question 33

What happens during the SOS response?

Answer 33

Synthesis of RecA is caused by DNA damage

1) Damaged single stranded DNA binds to RecA protein= Activates its protease function
2) RecA protein cleaves LexA protein (and also cI) to activate the SOS response
3) Cleavage of LexA removes repression of a number of SOS genes

If RecA can destroy the lambda repressor: left and right genes can be expressed again

Question 34

After SOS response, how can lytic pathway be started?

Answer 34

Transcription can now start from pL and pR again= Transcription in both directions

Excisase and integrase proteins made to remove the prophage from bacterial genome= Enters lytic pathway

Rightward operon= Everything needed for lytic pathway can be transcribed

Any attempt by cIII and cII to make more cI protein= FAIL= RecA protein will inactive it