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BIOC2001 CH > Bacteriophage and Bacteriophage Lambda > Flashcards

Bacteriophage and Bacteriophage Lambda Flashcards

1
Q

How many hydrogen bonds form between G-C and A-T?

A

G-C has 3, A-T has 2

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2
Q

Between which atoms do the hydrogen bonds of G and C form?

A

Of G: O6, N1, N2. At C: N4, N3, O2

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3
Q

Between which atoms do the hydrogen bonds of A and T form?

A

Of A: N6, N1. At T: O4, N3

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4
Q

What is the difference between a nucleotide and a nucleoside?

A

Nucleoside is base + sugar, nucleotide is base + sugar + phosphate

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5
Q

During DNA polymerisation, which groups join together (3’/5’ and functional group)

A

3’OH joins to 5’P

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6
Q

What is Chargaff’s rule?

A

The number of A/T and G/C is the same, therefore A must bond T and G bonds c

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7
Q

What is the difference between an endonuclease and an exonuclease?

A

Endonuclease cleaves DNA at multiple points internally, exonuclease cleaves sequentially (5’-3’) and more precisely

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8
Q

Where do restriction endonucleases originate from and what is important about their restriction sites?

A

Bacterial origin, palindromic recognition sites

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9
Q

Give an example of a restriction endonuclease which cuts to produce DNA with: 5’ overhang, 3’ overhand, no overhang

A

5’ - EcoR1, 3’-Pst1, none-Hpa1

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10
Q

What three things are required for ligation?

A

5’P, 3’OH and DNA ligase

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11
Q

What two types of ligation are there?

A

Sticky and blunt end

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12
Q

What is the mechanism of ligation (3 stages)?

A
  1. Adenylation of ligase (ATP/NAD addition causes AMP to bind to lysine)
  2. Activation of 5’P (AMP from ligase transferred to P in nick)
  3. Displacement of AMP (seals nick)
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13
Q

What does alkaline phosphatase do and what may it be used for?

A

Removes 5’P, can be used to prevent self-ligation (prevents ligation)

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14
Q

What is hyperchromicity?

A

The process of increase in A260 of DNA when it becomes single stranded

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15
Q

What is the role of topoisomerase type I and what is it’s method?

A

Uncoils DNA (nicks single strand, passes through loop and religates)

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16
Q

What is ethidium bromide used for and why?

A

Used as a fluorescent dye for DNA, it intercalates between DNA bases and fluoresces when exposed to UV light

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17
Q

What three types of bacteriophage are there?

A

Icosahedral w/ and w/out tails, filamentous

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18
Q

What are the 3 different types of genetic material a bacteriophage may have?

A

DNA/RNA, single/double stranded, circular/linear

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19
Q

What are the general life cycle stages of a bacteriophage?

A
  1. Initial phases - contacts cell, nucleic acid injected
  2. Early developmental - early genes expressed, nucleic acid replicated
  3. Late developmental - head/tail genes expressed, phage assembled
20
Q

In bacteriophage, what does virulent and temperate mean?

A

Virulent: phage will always follow the lytic pathway
Temperate: phage may follow lytic or lysogenic pathway depending on conditions

21
Q

What is a lysogen?

A

A bacteria cell with phage genome incorporated

22
Q

What is a prophage?

A

Phage DNA incorporated into bacterial chromosome (occurs during lysogeny)

23
Q

What is an induction event?

A

The process of excision of the phage genome from the host genome, the phage will follow the lytic cycle

24
Q

In temperate phage what are the two things which will decide whether the phage undergoes the lytic or lysogenic pathway?

A

Conditions: If there are many nutrients/little competition for the cells then the phage will enter the lytic cycle
Multiplicity of infection: high MoI (viruses»>bacteria) causes lysogenic pathway to be entered

25
Q

What method is used to determine whether phage are in lytic or lysogenic pathway and how?

A

Plaque assay: clear spots = lytic, turbid = lysogenic. Can determine whether phage are temperate or virulent also

26
Q

What form does bacteriophage lambda undertake and what is the form of it’s genetic material?

A

Icosahedral head w/ tail, linear, double stranded DNA

27
Q

How does the genetic material of bacteriophage lambda vary as it enters host cells?

A

The nucleic acid circularises (goes from linear to circular)

28
Q

What are the 3 phases involved in the lytic cycle development of bacteriophage lambda and what occurs in these phases? (do not mention the genes/promoters)

A

Early phase: regulator genes expressed -> new RNA polymerase made, sigma factor and anti-termination factor
Delayed early phase: replication enzymes, 2nd regulator
Late phase: structural phage components, cell lysis stuff

29
Q

What are the three phases involved in lytic cycle gene transcription (focus on promoters, genes, proteins produced and their effects)?

A

Immediate early: 2 promoter PR and PL are recognised by host cell transcriptional machinery and transcription of cro (PR) and N (PL) begins, tL1 and tR1 terminate transcription so only cro and pN are produced
Delayed early: pN anti-terminates (by acting on nut sites) causing transcription of Q, cII, cIII, 2xreplication, 5xrecombination and 2xDNA integrases
Late: initiates at PR’, pQ permits it to transcribe late genes (head & tail) lytic cycle begins

30
Q

What genes in the control region are responsible for the lytic pathway or the lysogenic pathway?

A

Lytic: N, cro, Q
Lysogenic: cIII, N, cI, cII

31
Q

What genes are present in the immunity region of bacteriophage lambda?

A

cIII –> cII (cIII, N, cI, cro, cII)

32
Q

Why does superinfection of a host cell not usually occur when phage are in lysogenic state? How may superinfection occur?

A

If a new phage with the same lambda repressor as the original phage attempts to infect then the original lambda repressors would bind to the new phage’s OR/OL preventing it from tending lytic cycle

33
Q

Describe the structure of the lambda repressor

A

27kDa, 2 domains (N & C), linker region –> dimer

34
Q

What are the N and C terminals of the lambda repressors responsible for?

A

N: forms oligomers. C: binds operators

35
Q

What promoters are responsible for cI expression?

A

PRM and PRE

36
Q

How does the lambda repressor affect OL and OR?

A

OL: binds and prevents PL from initiating transcription of N
OR: binds and prevents PR from initiating transcription of cro, also stimulates PRM

37
Q

How many alpha helices are present in the N-terminal domain of the lambda repressor and what are the roles of alpha 3 and 2?

A

5 alpha helices, 3 fits into major groove of DNA, 2 lies across major grove and H bonds to phosphate backbone

38
Q

How many binding sites does each operator (OL/OR) have?

A

3

39
Q

At regular lysogenic concentrations, which operator regions would have lambda repressor bound?

A

O1 and O2 (of both R and L)

40
Q

How may the shape of the lambda repressor be described?

A

Dumbbell shaped

41
Q

How does the binding of the lambda repressor to OR2 affect cI gene transcription?

A

When bound to OR2 the cI gene is transcribed due to interaction of lambda with PRM to enable more efficient binding of RNA polymerase

42
Q

Give an example of a cooperative interaction that takes place involving OR/OL

A

When lambda repressor binds OR1, OR2, OL1, OL2 it forms an octamer between these, bending the DNA strand round, this stabilises the operators therefore a 3x lower lambda repressor concentration is required for O1, O2 binding

43
Q

What happens if the lambda repressor concentration increases so that OR3 and OL3 are bound?

A

Binding to O3 would prevent expression of cI by PRM by preventing RNA pol binding

44
Q

What are the roles of cII and cIII in lysogeny?

A

cII and cIII are responsible for the initial synthesis of the lambda repressor: cII binds to PRE allowing RNA polymerase to bind, cIII protects cII from degradation

45
Q

How may a variation of cro transcription promote lysogeny?

A

cro may be transcribed the wrong way to create an antisense strand which may then bind to cro mRNA and inhibit expression

46
Q

What 4 events are required for lysogeny?

A
  1. cIII & cII transcription (cII activates PRE to make lambda repressor)
  2. lambda binds to PR & PL & stops all gene transcription (PRE also stops as no cII/cIII)
  3. lambda binds to O1 & O2, activates PRM
  4. Before cII is switched off it turns on Pi/Panti-Q promoters which transcribe int/antisense Q, respectively
47
Q

What is the role of cro and how does it achieve it’s role?

A

cro repressor prevents synthesis of lambda repressor by initially binding to OR3 (inactivating PRM), and then binding to OR1 & OR2, inactivating PR (and thus PRE - as no cII/cIII transcription)

BIOC2001 CH (3 decks)

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