Lecture 17 Flashcards
What are turbid plaques formed by?
Due to lysogens being immune to further infections
What did clear plaques indicate?
No lysogeny
What does CI ensure?
That no other phage can integrate. Gives the lysogen ‘immunity’
Which C protein maintains the prophage?
CI
CI is =
A repressor of all phage genes but an activator of itself. It keeps the phage genome
(Switching to lytic growth)
What are the two switch positions?
- Lysogeny = CI ON / Cro OFF (maintenance)
2. Lytic = CI OFF / Cro ON (induction)
What are the DNA components of the switch
- 3 operator sites
- 2 promotors
(Components of the switch - DNA)
Genes CI and Cro are transcribed ….
Divergently
(Components of the switch - DNA)
Between the genes, what sites are contained?
- Operator (binds CI and Cro)
2. Promotor (binds RNAP)
(Components of the switch - DNA)
How many operator sites are there for CI and Cro?
Three
-These overlap the promotors
(Components of the switch - DNA)
How many promotor sites are there for RNAP? Do these promotors overlap?
- Two
1. Prm (lysogenic)
2. Pr (lytic) - No they don’t overlap
(Components of the switch - RNAP)
What sites will RNAP bind to?
To either Pr or Prm, but never both
(Components of the switch - RNAP)
What type of promotor is Pr and describe its relationship to regulatory proteins
It is a strong promotor and therefore doesn’t require regulatory proteins
(Components of the switch - RNAP)
If there are no regulatory proteins around, where will RNAP bind to?
Pr
-Shows the default pathway is in this direction (lytic)
(Components of the switch - RNAP)
What type of promotor is Prm and describe its relationship to regulatory proteins
It needs CI as an activator due to its weakness as a promotor
Are operator sites identical?
No, they are similar but not identical so CI and Cro can distinguish between them. This is due to different affinities and binding
Is protein binding to DNA reversible?
Yes
What can affinities determine the order of?
Determine the order of binding relative to protein concentration
(Components - CI)
In lysogenic cells, what percentage of CI is dimeric and by what interactions?
- 95%
- Via C-terminal interactions
(Components - CI)
How is the dimer formed?
Through 2 interactions
- N terminal = interacts with DNA, binding to operator sites
- C terminal = forms dimer by protein-protein interactions
(Components - CI)
How many dimers can each Or site bind?
One CI dimer
What are the two functions of CI?
- Negative Control
2. Positive control
Describe the CI function of negative control
At Or2, CI turns off Cro by preventing RNAP from binding to Cro promotor (exclusion)
Describe the CI function of positive control
At Or2, CI helps RNAP bind and begins transcription of CI gene
- > RNAP would usually be unstable at this site. CI facilitates protein-protein interactions which enable more RNAPol to bind to the promoter. The interactions stabilise the RNAPol.
- > Increases transcription
Which operator sites does CI prefer?
CI prefers operator sites 1 and 2 and binds to these sites with a higher affinity than OR3
What occurs when CI binds to Or1?
Switches off the lytic pathway (lysogenic on)
What occurs when CI binds to Or3?
Lytic growth occurs, CI is OFF due to RNAP exclusion
What combination of Or’s are most likely to be switched on in a lambda lysogen?
Or1 and 2
What two factors contribute to CI binding?
- Intrinsic affinity for binding sites
2. Cooperativity
What will CI binding at Or1 site cause for Or2?
- Binding at Or1 gives increased affinity for Or2
- >this requires protein-protein interactions
Describe CI binding at Or3
- Binding here is weak with no cooperativity
- The protein heads turn away from each other
- Only binds to this sport when CI concentrations are high
Describe the maintenance of lysogeny through Or’s
- CI bound at Or1 and Or2 inhibits Cro and maintains expression of CI
- This is a very stable state which is inherited by daughter cells following cell division
What occurs to flip the switch from lysogeny to lytic pathway?
-Stresses like UV can trigger induction
Describe the effect of UV damage on induction
- UV damage causes RecA to bind to CI and stimulate an auto-proteolitic process to cleave itself
- This clears the operator sites of CI and deems CI as two nonfunctional domains
What is the result of CI cleavage?
- Activation of Prm (CI) is lost
- Repression of excision gene is lost (decrease CI)
- RNAP binds to Pr and transcribes Cro
What are the features of Cro?
- Cro can bind to each Or site in absence of CI
- Cro has a single domain
- Most of Cro in the cell is dimeric
How does Cro bind to each operator site?
- Binds independently
- As Cro concentration increases, it starts to bind to the sites with relatively equal affinity. There is NO COOPERATIVITY INVOLVED
What kind of regulator is Cro?
Negative
What Or site does Cro prefer to bind on?
Or3
When the particles want to excise from the host, how does the CI cleavage help this occur?
- Following the CI cleavage, expression of Pint is lost
- Int and Xis proteins are make which the combination of these promote excision of lambda from the host
