7. Bacteriophages 1 Flashcards
What is the unique thing about MS2 phage?
protein translation
what is the unique thing about phiX174 phage?
rolling circle replication of DNA
what is the unique thing about T7 phage?
transcription coordinated with entry
what is the unique thing about lambda phage?
prophage (integrates phage DNA into bacterial host DNA) and complex regulation of gene expression
which domains of life are infected by viruses?
all 3! bacteria, archaea, and eukaryotes
what are the 3 similarities btwn bacteria and archaea?
- unicellular
- circular DNA
- no nucleus
what are 9 unique characteristics that archaea have but bacteria don’t?
- cell walls made of impermeable S layer proteins
- special phospholipids
- translation initiation mimics eukaryotes
- uses methionine (not N-formyl methionine) to start translation
- histone-like proteins to package chromosomes
- complex RNA pol machinery
- DNA replication machinery mimics eukaryotes
- distinct rRNA sequences
- live in extreme environments
what is the morphology of viruses of archaea?
UNUSUAL!
- lemon
- droplet
- bottle
etc
what type of genomes do viruses of archaea have?
dsDNA except 1 has ssDNA
describe the envelopes of viruses of archaea?
MOST have internal or external lipid envelopes
what does it mean for viruses of archaea to be temperate?
temperate = LATENT –> they integrate their genome into host cell DNA without killing the cell
describe the DNA polymerase of viruses of archaea?
many don’t have identifiable DNA polymerase gene
what virus is this?
sulfolobus spindle-shaped virus 1 (SSV1)
what virus is this?
acidianus two tailed virus (ATV)
what virus is this?
acidianus bottle-shaped virus (ABV)
what virus is this?
sulfolobus neozealandicus droplet-shaped virus (SNDV)
why do we not need to worry about viruses of archaea?
we don’t go to the extreme environments where archaea live
when were phages discovered? by who?
Twort and d’Herelle discovered them in 1915/1917
when were phages first visualized?
in 1940
what was found in 1969? by who?
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
what was found in 1978? by who?
Arber, Nathans, and Smith discovered restriction enzymes and how they could be used to open a vector and insert DNA for cloning
how do bacteria use restriction enzymes? explain
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
when were viruses sequenced? what type of tools were used + 3 examples?
MS2 - 1976
phiX174 - 1977
lambda - 1982 using PHAGE-DERIVED TOOLS –> restriction enzymes, T4 DNA ligase, M13 vectors
how do we use phage-derived tools?
to sequence everything
what was found in the 1990s?
there is an abundance of phages in our ecosystem that have co-evolved with bacteria via gene transfer
besides restriction enzymes, what is another defense mechanism of bacteria against phages? how does it work?
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
how do we use CRISPR in labs?
to edit genes
what is phage therapy used for?
to target bacterial infection
where does the word bacteriophage come from?
bacteria + phagein (to devour)
what type of molecule are bacteriophages?
obligate intracellular parasites
which type of bacteria do bacteriophages infect?
all natural populations of bacteria
what are the most widely distributed and diverse entities in the biosphere? how many
bacteriophages –> 10^31
do all bacteriophages have the same structure?
no
what is the structure of T4 phages?
head, contractile tail, long fibers
what is the structure of lambda phages?
head, long non-contractile flexible tail, short fibers
what is the structure of T7 phages?
no tail, short fibers, head
what is the structure of phiX174 phages?
only head
What is the structure of M13 phage?
no head only fiber (ebola similar shape)
what are the 3 possible type of genomes in bacteriophages?
- ssRNA
- ssDNA
- dsDNA
what are the 2 families of ssRNA bacteriophages? and examples of each
- LEVIviridae –> MS2
- INOviridae –> f1, fd, M13
what is the family of ssDNA bacteriophages? and an example
MICROviridae –> phiX174
what are the 2 families of dsDNA bacteriophages? and examples of each
- PODOviridae –> T7
- SIPHOviridae –> lambda
what is unique about dsDNA bacteriophages?
they have tails
do bacteriophages have envelopes?
no
describe MS2 genome
(+) ssRNA, 4kb
how many maturation proteins are in the MS2 phage?
1
how many coat proteins are in the MS2 phage?
180 copies
describe MS2 entry and lifecycle in bacterial cell (5 steps)
- virus binds to sex pilus
- viral RNA enters the cell
- ssRNA translates into phage proteins
- ssRNA: (+) –> (-) to make more (+)
- assembly
how many viral phage genes are in MS2? what are they?
- maturation
- coat
- lysis
- replicase
what proteins are in the MS2 viral particle? which are not?
maturation and coat protein in MS2 particle
lysis and replicase are not
what are the 5 general steps of MS2 protein synthesis?
- coat protein produced upon entry
- allows replicase and lysis proteins to be produced
- replicase binds to start codon of coat gene to block coat protein synthesis, copies (+) RNA –> (-) –> (+) to make more genome
- the new (+) RNA folds to allow synthesis of maturation protein
- MORE coat proteins produced and form dimers that stop replicase production and initiate phage assembly
what is the role of replicase protein?
amplify phage genome
what is the role of MS2 coat protein on 1st production and 2nd production?
1st: allows production of replicase and lysis proteins
2nd: shut down replicase protein and initiate assembly of phage particles
how does the production of MST coat protein allow for production of replicase and lysis proteins?
coat protein start codon is easily accessible but lysis and replicase start codons are hidden:
- host ribosomes can recognize start codon of coat protein to translate it
- once coat is translated, lysis and replicase hairpins open and expose their start codons
how does folding of RNA regulate the translation of maturation protein?
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
what does replicase do?
what helps replicase do its job?
replicase binds to the start of the coat gene to shut down coat protein translation
replicase is associated with 3 HOST proteins
what are the 3 host proteins associated with replicase? and what do they each do?
- S1 protein in the small ribosomal subunit –> directs replicase to the start of coat gene
- EF-Tu translational elongation factor –> with GTP, helps replicase initiate RNA synthesis
- EF-Ts translational elongation factor –> recycles EF-Tu/GDP to EF-Tu/GTP
how do we use MS2/RNA binding to monitor RNA?
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
what type of virus is phiX174?
microvirus
what type of genome does phiX174 have?
ssDNA
does phiX174 have its own polymerase?
no –> uses host polymerase
describe phiX174 entry into the cell (2 steps)
- capsid interacts with sugar residues (ex. glucose) in LPS of bacteria
- H protein in spike penetrates cell wall then forms channel for DNA to be delivered thru viral spikes
what proteins are in phiX174 spikes?
G and H proteins
what allows H protein to mediate the penetration step?
H protein has an N-terminal transmembrane helix
what is the first stage of phiX174 genome replication?
circular (+) ssDNA uses host proteins to start DNA synthesis with primosome –> host DNA pol makes circular dsDNA
what is the second stage of phiX174 genome replication?
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
how many promoters does the phiX174 genome use?
3
how many terminators does the phiX174 genome use?
4
what 4 factors control viral gene expression of phiX174?
- transcription strength of promoters
- stability of viral RNA
- termination efficiency of terminators
- ribosome binding site
if there are only 3 promoters, how can phiX174 express more than 3 genes?
promoter stops at different sites to control the amount of mRNA and therefore the amount of protein
what are the 2 scaffolding proteins expressed by phiX174? what is their role for phiX174 phage?
protein B and protein D
they help with assembly, then removed by protease to allow complete maturation and become infectious
why is a provirion not infectious?
even though it has all its proteins, a provirion still has scaffolding proteins
what does the assembly of phiX174 start with?
starts with assembled units, not monomer
