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GENE221 > Lecture 14 > Flashcards

Lecture 14 Flashcards

1
Q

Lysogeny are immune to..

A

Other bacteriophage

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2
Q

Why does lambda form turbid plaques

A

Lambda forms turbid plaques due to lysogeny being immune to further infections - growth in a plaque

Sometimes clear plaques were observed (no lysogeny), mutants named cI, cII and cIII ( clear mutants)

Turbid - cloudy plaques due to lytic and lysogenic pathways

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3
Q

CI protein is both a repressor of all the phage genes but an activator of itself

A

Yep

Means there will always be enough of it present to be silencing the phage genome

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4
Q

The role of CI in the maintancance of lysogeny

A
  • lambda is maintained as a prophage in the lysogen by CI
  • CI is a repressor of all phage genes but an activator of itself
  • keeps phage genome ‘silent’ in bacterial chromosome
  • repressor protects lysogen from further lambda infection via ‘immunity’
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5
Q

The switch to lytic growth - overview

A
  • UV irradiation inactivates CI
  • another protein, Cro, synthesised and lytic growth occurs
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6
Q

Basic components of the lytic growth switch

A
  • regulatory proteins CI (repressor) and Cro
  • operator sites on the phage DNA and their promoters
  • RNA polymerase (essential for transcription
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7
Q

The two “switch” positions of lytic growth

A

1: lysogeny: CI ON and Cro OFF (Maintenance)
2. Lytic: CI OF and Cro ON (Induction)

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8
Q

promoter region between CI and Cro 0 (region where Cro is being transcribed in one direction and CI in the other) - what is in this region

A

Region (80bp) between genes contain two types of sites:
1. Operator (binds CI and Cro)
2. Promoter (binds RNAP)

Three operator sites (OR1, OR2 and OR3) for CI and Cro

Two promoters for RNAP (PRM and PR) - do not overlap

(The R stands for right hand region)

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9
Q

The role of RNAP in the “switch”

A

RNAP (RNA polymerase) transcribes DNA into RNA and is provided by the bacterial host

RNAP will bind to wither PR or PRM but never both

PR activity by RNAP does not require regulatory protein, whereas PRM needs CL as an activator

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10
Q

What happens when RNAP binds to PRM

A

Cl is turned ON

PRM M for MAINTAIN

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11
Q

What happens when RNAP binds to PR

A

Turns on Cro

  • this is the default pathway whereas PRM needs an activator to help expression
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12
Q

Features of operator sites

A
  • operator sites overlap either promoters (e.g: OR1 And OR3) or both promoters (e.g: OR2)
  • operator sites are 17 bp and similar but not identical so CI and Cro can distinguish between them (different affinities)
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13
Q

Profiting binding to DNA is ____

A

Reversible

  • some regulator binds and come falls off
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14
Q

Low binding protein vs high binding protein

A
  • first occupy the really strong sites then the weaker sites
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15
Q

Components of the switch that allow CI to act as a repressor

A
  • in a lysogenic cell ~95% of CI is dimeric via c-terminal interations
  • CI diners use their N-terminal domain to bind DNA
  • each OR sire can bind one CI dimer along one side of the DNA helix
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16
Q

How does CI work? - the two functions

A

Negative control: CI at OR2 turns off Cro gene by preventing RNAP from binding to Cro promoter (exclusion)

Positive control: Cl at OR2 helps RNAP bind and begin transcription of CI gene (10 X upregualtion). (This is cos Yk, assists the weak binding??)

CI increases RNAP for PRM by providing protein-protein interations in addition to the protein-DNA interations

17
Q

Which operator site does CI prefer?

A
18
Q

Two functions of CI diagram

A
19
Q

What happens if Cl were to bind to OR1 (but it can’t)

A

CI off since no activation and Cro off due to exclusion
- no life cycle

20
Q

What happens if CI binds to OR3

A

Cl off due to RNAP exclusion and Cro on
- lytic

21
Q

What’s up with OR1 and OR2 in lambda lysogeny

A

They are usually bound by CI (90% of cells)
(CI on and Cro off)
- this maintains the lysogeny

Sometimes CI is bound to OR3 too (less then 10%)
- switches off it’s own production
- both are off
- of there are too much CI the cell turns off CI production until cell divides and levels of CI drop again

22
Q

Two factors that contribute to CI binding

A

Intrinsic affinity
Cooperativity

23
Q

Cooperativity and affinity of the OR sites

A

Bound CI at OR1 increases affinity for OR2 (aided by protein-protein interactions)

Binding at OR3 weak and no Cooperativity

24
Q

Cooperativity of CI binding at the 3 sites - how it works

A
  • dimers of CI ‘lean’ towards eachother
  • CI binding at OR3 cannot be cooperative since CI C-terminal domains unavailable
  • CI binding at OR2 can still bind RNAP since it interacts with N-terminus
25
Q

Maintaince of lysogeny - CI postiotns

A

In lysogeny CI bound at OR1 and OR2 inhibits Cro and maintains exporession of CI

This is very stable and this state is inherited by daughter cells after cell division (epigenetic) - lots of CI inherited each time the cell divides

26
Q

Process of induction

A

UV induction can cause prophage to enter lytic cycle

  1. Prophage excises from the host chromosome
  2. Lytic growth
27
Q

What does induction via UV cause

A

Results in DNA damage that is somehow sensed by RecA resulting in it becoming a co-protease

Activated by RecA assists CI cleavage into two domains (non-functional) and therefore clears the operator sites of CI

28
Q

Results of the CI cleavage after flipping the switch

A
  1. Activation of PRM (cI) lost
  2. Repression of excision genes lost (decreased CI)
  3. RNAP binds to PR and transcribes Cro
  4. Cro now determines the course of events
29
Q

Features of Cro - in the switch

A
  • Cro has a single dominant
  • two monomers of Cro form dimers
  • almost all Cro in the cell is dimeric
  • Cro dimers can binds to each OR site in absence of CI
  • Cro binds on one side of the DNA helix

CI AND CRO BIND SMAE OR SIRES BUT HAVE OPPOSITE EFFECTS

30
Q

Induction - the features of Cro

A
  • once some Cro is produced it will bind to the highest affinity site (OR3) - oposite one of CI - afterwards OR1
  • as more protein is produced CRO will bind all the sites
  • Cro binds to each operator site indiepentily
  • Cro is only a NEGATIVE regulator
  • binding of Cro to operators is roughly opposite that of CI
31
Q

Induction - the effect of Cro

A

Ceebs making cue card

32
Q

Process of switches excision from host

A

Results of CI cleavage:
- lose repression of PINT and the int and Xis proteins are made
- Xis/int promote excision of lambda from host

33
Q
A

GENE221 (23 decks)

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