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GENE221 > Lecture 17 > Flashcards

Lecture 17 Flashcards

1
Q

What are turbid plaques formed by?

A

Due to lysogens being immune to further infections

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2
Q

What did clear plaques indicate?

A

No lysogeny

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3
Q

What does CI ensure?

A

That no other phage can integrate. Gives the lysogen ‘immunity’

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4
Q

Which C protein maintains the prophage?

A

CI

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5
Q

CI is =

A

A repressor of all phage genes but an activator of itself. It keeps the phage genome

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6
Q

(Switching to lytic growth)

What are the two switch positions?

A
  1. Lysogeny = CI ON / Cro OFF (maintenance)

2. Lytic = CI OFF / Cro ON (induction)

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7
Q

What are the DNA components of the switch

A
  • 3 operator sites

- 2 promotors

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8
Q

(Components of the switch - DNA)

Genes CI and Cro are transcribed ….

A

Divergently

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9
Q

(Components of the switch - DNA)

Between the genes, what sites are contained?

A
  1. Operator (binds CI and Cro)

2. Promotor (binds RNAP)

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10
Q

(Components of the switch - DNA)

How many operator sites are there for CI and Cro?

A

Three

-These overlap the promotors

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11
Q

(Components of the switch - DNA)

How many promotor sites are there for RNAP? Do these promotors overlap?

A
  • Two
    1. Prm (lysogenic)
    2. Pr (lytic)
  • No they don’t overlap
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12
Q

(Components of the switch - RNAP)

What sites will RNAP bind to?

A

To either Pr or Prm, but never both

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13
Q

(Components of the switch - RNAP)

What type of promotor is Pr and describe its relationship to regulatory proteins

A

It is a strong promotor and therefore doesn’t require regulatory proteins

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14
Q

(Components of the switch - RNAP)

If there are no regulatory proteins around, where will RNAP bind to?

A

Pr

-Shows the default pathway is in this direction (lytic)

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15
Q

(Components of the switch - RNAP)

What type of promotor is Prm and describe its relationship to regulatory proteins

A

It needs CI as an activator due to its weakness as a promotor

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16
Q

Are operator sites identical?

A

No, they are similar but not identical so CI and Cro can distinguish between them. This is due to different affinities and binding

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17
Q

Is protein binding to DNA reversible?

A

Yes

18
Q

What can affinities determine the order of?

A

Determine the order of binding relative to protein concentration

19
Q

(Components - CI)

In lysogenic cells, what percentage of CI is dimeric and by what interactions?

A
  • 95%

- Via C-terminal interactions

20
Q

(Components - CI)

How is the dimer formed?

A

Through 2 interactions

  1. N terminal = interacts with DNA, binding to operator sites
  2. C terminal = forms dimer by protein-protein interactions
21
Q

(Components - CI)

How many dimers can each Or site bind?

A

One CI dimer

22
Q

What are the two functions of CI?

A
  1. Negative Control

2. Positive control

23
Q

Describe the CI function of negative control

A

At Or2, CI turns off Cro by preventing RNAP from binding to Cro promotor (exclusion)

24
Q

Describe the CI function of positive control

A

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
25
Q

Which operator sites does CI prefer?

A

CI prefers operator sites 1 and 2 and binds to these sites with a higher affinity than OR3

26
Q

What occurs when CI binds to Or1?

A

Switches off the lytic pathway (lysogenic on)

27
Q

What occurs when CI binds to Or3?

A

Lytic growth occurs, CI is OFF due to RNAP exclusion

28
Q

What combination of Or’s are most likely to be switched on in a lambda lysogen?

A

Or1 and 2

29
Q

What two factors contribute to CI binding?

A
  1. Intrinsic affinity for binding sites

2. Cooperativity

30
Q

What will CI binding at Or1 site cause for Or2?

A
  • Binding at Or1 gives increased affinity for Or2

- >this requires protein-protein interactions

31
Q

Describe CI binding at Or3

A
  • 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
32
Q

Describe the maintenance of lysogeny through Or’s

A
  • 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
33
Q

What occurs to flip the switch from lysogeny to lytic pathway?

A

-Stresses like UV can trigger induction

34
Q

Describe the effect of UV damage on induction

A
  • 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
35
Q

What is the result of CI cleavage?

A
  1. Activation of Prm (CI) is lost
  2. Repression of excision gene is lost (decrease CI)
  3. RNAP binds to Pr and transcribes Cro
36
Q

What are the features of Cro?

A
  • Cro can bind to each Or site in absence of CI
  • Cro has a single domain
  • Most of Cro in the cell is dimeric
37
Q

How does Cro bind to each operator site?

A
  • Binds independently
  • As Cro concentration increases, it starts to bind to the sites with relatively equal affinity. There is NO COOPERATIVITY INVOLVED
38
Q

What kind of regulator is Cro?

A

Negative

39
Q

What Or site does Cro prefer to bind on?

A

Or3

40
Q

When the particles want to excise from the host, how does the CI cleavage help this occur?

A
  • 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

GENE221 (29 decks)

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