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MIMM 324 > 7. Bacteriophages 1 > Flashcards

7. Bacteriophages 1 Flashcards

1
Q

What is the unique thing about MS2 phage?

A

protein translation

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2
Q

what is the unique thing about phiX174 phage?

A

rolling circle replication of DNA

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3
Q

what is the unique thing about T7 phage?

A

transcription coordinated with entry

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4
Q

what is the unique thing about lambda phage?

A

prophage (integrates phage DNA into bacterial host DNA) and complex regulation of gene expression

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5
Q

which domains of life are infected by viruses?

A

all 3! bacteria, archaea, and eukaryotes

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6
Q

what are the 3 similarities btwn bacteria and archaea?

A
  1. unicellular
  2. circular DNA
  3. no nucleus
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7
Q

what are 9 unique characteristics that archaea have but bacteria don’t?

A
  1. cell walls made of impermeable S layer proteins
  2. special phospholipids
  3. translation initiation mimics eukaryotes
  4. uses methionine (not N-formyl methionine) to start translation
  5. histone-like proteins to package chromosomes
  6. complex RNA pol machinery
  7. DNA replication machinery mimics eukaryotes
  8. distinct rRNA sequences
  9. live in extreme environments
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8
Q

what is the morphology of viruses of archaea?

A

UNUSUAL!
- lemon
- droplet
- bottle
etc

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9
Q

what type of genomes do viruses of archaea have?

A

dsDNA except 1 has ssDNA

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10
Q

describe the envelopes of viruses of archaea?

A

MOST have internal or external lipid envelopes

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11
Q

what does it mean for viruses of archaea to be temperate?

A

temperate = LATENT –> they integrate their genome into host cell DNA without killing the cell

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12
Q

describe the DNA polymerase of viruses of archaea?

A

many don’t have identifiable DNA polymerase gene

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13
Q

what virus is this?

A

sulfolobus spindle-shaped virus 1 (SSV1)

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14
Q

what virus is this?

A

acidianus two tailed virus (ATV)

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15
Q

what virus is this?

A

acidianus bottle-shaped virus (ABV)

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16
Q

what virus is this?

A

sulfolobus neozealandicus droplet-shaped virus (SNDV)

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17
Q

why do we not need to worry about viruses of archaea?

A

we don’t go to the extreme environments where archaea live

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18
Q

when were phages discovered? by who?

A

Twort and d’Herelle discovered them in 1915/1917

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19
Q

when were phages first visualized?

A

in 1940

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20
Q

what was found in 1969? by who?

A

Delbruck, Hershey, and Luria found how phages replicate DNA and express genes

i.e. using phages, found that DNA was genetic material bc DNA alone could dictate production of new phages

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21
Q

what was found in 1978? by who?

A

Arber, Nathans, and Smith discovered restriction enzymes and how they could be used to open a vector and insert DNA for cloning

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22
Q

how do bacteria use restriction enzymes? explain

A

bacteria use them as a defense mechanism against phages

restriction enzymes target a small DNA sequence –> this sequence is modified in bacteria but remains the same in phages so the enzymes can cleave phage DNA

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23
Q

when were viruses sequenced? what type of tools were used + 3 examples?

A

MS2 - 1976
phiX174 - 1977
lambda - 1982 using PHAGE-DERIVED TOOLS –> restriction enzymes, T4 DNA ligase, M13 vectors

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24
Q

how do we use phage-derived tools?

A

to sequence everything

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25
Q

what was found in the 1990s?

A

there is an abundance of phages in our ecosystem that have co-evolved with bacteria via gene transfer

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26
Q

besides restriction enzymes, what is another defense mechanism of bacteria against phages? how does it work?

A

CRISPR

acts as bacterial immune system memory –> phage DNA is inserted into host DNA so the bacteria can remember which phages were there to attack upon next infection

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27
Q

how do we use CRISPR in labs?

A

to edit genes

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28
Q

what is phage therapy used for?

A

to target bacterial infection

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29
Q

where does the word bacteriophage come from?

A

bacteria + phagein (to devour)

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30
Q

what type of molecule are bacteriophages?

A

obligate intracellular parasites

31
Q

which type of bacteria do bacteriophages infect?

A

all natural populations of bacteria

32
Q

what are the most widely distributed and diverse entities in the biosphere? how many

A

bacteriophages –> 10^31

33
Q

do all bacteriophages have the same structure?

A

no

34
Q

what is the structure of T4 phages?

A

head, contractile tail, long fibers

35
Q

what is the structure of lambda phages?

A

head, long non-contractile flexible tail, short fibers

36
Q

what is the structure of T7 phages?

A

no tail, short fibers, head

37
Q

what is the structure of phiX174 phages?

A

only head

38
Q

What is the structure of M13 phage?

A

no head only fiber (ebola similar shape)

39
Q

what are the 3 possible type of genomes in bacteriophages?

A
  1. ssRNA
  2. ssDNA
  3. dsDNA
40
Q

what are the 2 families of ssRNA bacteriophages? and examples of each

A
  1. LEVIviridae –> MS2
  2. INOviridae –> f1, fd, M13
41
Q

what is the family of ssDNA bacteriophages? and an example

A

MICROviridae –> phiX174

42
Q

what are the 2 families of dsDNA bacteriophages? and examples of each

A
  1. PODOviridae –> T7
  2. SIPHOviridae –> lambda
43
Q

what is unique about dsDNA bacteriophages?

A

they have tails

44
Q

do bacteriophages have envelopes?

A

no

45
Q

describe MS2 genome

A

(+) ssRNA, 4kb

46
Q

how many maturation proteins are in the MS2 phage?

A

1

47
Q

how many coat proteins are in the MS2 phage?

A

180 copies

48
Q

describe MS2 entry and lifecycle in bacterial cell (5 steps)

A
  1. virus binds to sex pilus
  2. viral RNA enters the cell
  3. ssRNA translates into phage proteins
  4. ssRNA: (+) –> (-) to make more (+)
  5. assembly
49
Q

how many viral phage genes are in MS2? what are they?

A
  1. maturation
  2. coat
  3. lysis
  4. replicase
50
Q

what proteins are in the MS2 viral particle? which are not?

A

maturation and coat protein in MS2 particle

lysis and replicase are not

51
Q

what are the 5 general steps of MS2 protein synthesis?

A
  1. coat protein produced upon entry
  2. allows replicase and lysis proteins to be produced
  3. replicase binds to start codon of coat gene to block coat protein synthesis, copies (+) RNA –> (-) –> (+) to make more genome
  4. the new (+) RNA folds to allow synthesis of maturation protein
  5. MORE coat proteins produced and form dimers that stop replicase production and initiate phage assembly
52
Q

what is the role of replicase protein?

A

amplify phage genome

53
Q

what is the role of MS2 coat protein on 1st production and 2nd production?

A

1st: allows production of replicase and lysis proteins

2nd: shut down replicase protein and initiate assembly of phage particles

54
Q

how does the production of MST coat protein allow for production of replicase and lysis proteins?

A

coat protein start codon is easily accessible but lysis and replicase start codons are hidden:

  1. host ribosomes can recognize start codon of coat protein to translate it
  2. once coat is translated, lysis and replicase hairpins open and expose their start codons
55
Q

how does folding of RNA regulate the translation of maturation protein?

A

when the RNA is folding up, it has an intermediate structure for a short time where the ribosome can access the start codon and make a few maturation protein

then the mature RNA is folded up, hiding the ribosome entry site for the maturation protein

56
Q

what does replicase do?
what helps replicase do its job?

A

replicase binds to the start of the coat gene to shut down coat protein translation

replicase is associated with 3 HOST proteins

57
Q

what are the 3 host proteins associated with replicase? and what do they each do?

A
  1. S1 protein in the small ribosomal subunit –> directs replicase to the start of coat gene
  2. EF-Tu translational elongation factor –> with GTP, helps replicase initiate RNA synthesis
  3. EF-Ts translational elongation factor –> recycles EF-Tu/GDP to EF-Tu/GTP
58
Q

how do we use MS2/RNA binding to monitor RNA?

A

add MS2 to 3’ end of gene and fuse with GFP

MS2 binds to RNA and when RNA is exported to the cytoplasm, GFP indicates where RNA is located

59
Q

what type of virus is phiX174?

A

microvirus

60
Q

what type of genome does phiX174 have?

A

ssDNA

61
Q

does phiX174 have its own polymerase?

A

no –> uses host polymerase

62
Q

describe phiX174 entry into the cell (2 steps)

A
  1. capsid interacts with sugar residues (ex. glucose) in LPS of bacteria
  2. H protein in spike penetrates cell wall then forms channel for DNA to be delivered thru viral spikes
63
Q

what proteins are in phiX174 spikes?

A

G and H proteins

64
Q

what allows H protein to mediate the penetration step?

A

H protein has an N-terminal transmembrane helix

65
Q

what is the first stage of phiX174 genome replication?

A

circular (+) ssDNA uses host proteins to start DNA synthesis with primosome –> host DNA pol makes circular dsDNA

66
Q

what is the second stage of phiX174 genome replication?

A

viral A protein cleaves circular dsDNA to make exposed 3’ end that acts as a primer to make ssDNA –> elongates –> new ssDNA is cleaved out and circularized to make new (+) ssDNA

67
Q

how many promoters does the phiX174 genome use?

A

3

68
Q

how many terminators does the phiX174 genome use?

A

4

69
Q

what 4 factors control viral gene expression of phiX174?

A
  1. transcription strength of promoters
  2. stability of viral RNA
  3. termination efficiency of terminators
  4. ribosome binding site
70
Q

if there are only 3 promoters, how can phiX174 express more than 3 genes?

A

promoter stops at different sites to control the amount of mRNA and therefore the amount of protein

71
Q

what are the 2 scaffolding proteins expressed by phiX174? what is their role for phiX174 phage?

A

protein B and protein D

they help with assembly, then removed by protease to allow complete maturation and become infectious

72
Q

why is a provirion not infectious?

A

even though it has all its proteins, a provirion still has scaffolding proteins

73
Q

what does the assembly of phiX174 start with?

A

starts with assembled units, not monomer

MIMM 324 (37 decks)

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