Lecture 17

Question 1

What are turbid plaques formed by?

Answer 1

Due to lysogens being immune to further infections

Question 2

What did clear plaques indicate?

Answer 2

No lysogeny

Question 3

What does CI ensure?

Answer 3

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

Question 4

Which C protein maintains the prophage?

Answer 4

CI

Question 5

CI is =

Answer 5

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

Question 6

(Switching to lytic growth)

What are the two switch positions?

Answer 6

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

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

Question 7

What are the DNA components of the switch

Answer 7

• 3 operator sites

- 2 promotors

Question 8

(Components of the switch - DNA)

Genes CI and Cro are transcribed ….

Answer 8

Divergently

Question 9

(Components of the switch - DNA)

Between the genes, what sites are contained?

Answer 9

1. Operator (binds CI and Cro)

2. Promotor (binds RNAP)

Question 10

(Components of the switch - DNA)

How many operator sites are there for CI and Cro?

Answer 10

Three

-These overlap the promotors

Question 11

(Components of the switch - DNA)

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

Answer 11

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

Question 12

(Components of the switch - RNAP)

What sites will RNAP bind to?

Answer 12

To either Pr or Prm, but never both

Question 13

(Components of the switch - RNAP)

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

Answer 13

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

Question 14

(Components of the switch - RNAP)

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

Answer 14

Pr

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

Question 15

(Components of the switch - RNAP)

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

Answer 15

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

Question 16

Are operator sites identical?

Answer 16

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

Question 17

Is protein binding to DNA reversible?

Answer 17

Yes

Question 18

What can affinities determine the order of?

Answer 18

Determine the order of binding relative to protein concentration

Question 19

(Components - CI)

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

Answer 19

• 95%

- Via C-terminal interactions

Question 20

(Components - CI)

How is the dimer formed?

Answer 20

Through 2 interactions

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

Question 21

(Components - CI)

How many dimers can each Or site bind?

Answer 21

One CI dimer

Question 22

What are the two functions of CI?

Answer 22

1. Negative Control

2. Positive control

Question 23

Describe the CI function of negative control

Answer 23

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

Question 24

Describe the CI function of positive control

Answer 24

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

Question 25

Which operator sites does CI prefer?

Answer 25

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

Question 26

What occurs when CI binds to Or1?

Answer 26

Switches off the lytic pathway (lysogenic on)

Question 27

What occurs when CI binds to Or3?

Answer 27

Lytic growth occurs, CI is OFF due to RNAP exclusion

Question 28

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

Answer 28

Or1 and 2

Question 29

What two factors contribute to CI binding?

Answer 29

1. Intrinsic affinity for binding sites | 2. Cooperativity

Question 30

What will CI binding at Or1 site cause for Or2?

Answer 30

- Binding at Or1 gives increased affinity for Or2 | - >this requires protein-protein interactions

Question 31

Describe CI binding at Or3

Answer 31

- 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

Question 32

Describe the maintenance of lysogeny through Or's

Answer 32

- 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

Question 33

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

Answer 33

-Stresses like UV can trigger induction

Question 34

Describe the effect of UV damage on induction

Answer 34

- 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

Question 35

What is the result of CI cleavage?

Answer 35

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

Question 36

What are the features of Cro?

Answer 36

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

Question 37

How does Cro bind to each operator site?

Answer 37

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

Question 38

What kind of regulator is Cro?

Answer 38

Negative

Question 39

What Or site does Cro prefer to bind on?

Answer 39

Or3

Question 40

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

Answer 40

- 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