8. Bacteriophages 2 Flashcards
what is the family of T7 phage?
podoviridae
what is the shape of T7 phage?
- icosahedral capsid
- short tail
- some tail fibers
what is the size of T7 phage?
60nm
is T7 lytic? is T7 lysogenic?
LYTIC, not lysogenic
what does it mean for a virus to be lytic but not lysogenic?
the virus infects and kills the cell but doesn’t integrate its DNA into the host DNA
describe the T7 genome:
- shape
- type of genome
- number of bp
- number of genes
- number of proteins
- LINEAR
- dsDNA
- 40kbp
- 56 genes
- 59 proteins
what is a unique feature of the T7 genome? what is the purpose?
5’ end and 3’ end have identical 150bp sequences (terminal repeats)
critical for replication of phage DNA
what is the receptor on bacteria that the phage interacts with?
LPS
what are the 2 parts of T7 replication that are unique?
- RNA transcription
- DNA replication
what are the 3 classes of proteins in the T7 genome?
- Class I –> early proteins
- Class II –> DNA metabolism/replication
- Class III –> virion structure and assembly
describe the organization of genes on the T7 genome
genes with the same function/involved in the same pathway are organized in clusters –> OPERONS
describe the promoters and terminators used in T7?
in each class, each gene is expressed at different promoters but tend to stop at the same terminators
describe the 5 steps for entry of T7 DNA into cytoplasm and beginning of transcription
- phage binds LPS on outer membrane
- internal core proteins (gp14,15,16) form a channel into cytoplasm to inject phage DNA
- 850bp phage DNA enters with help from gp16
- bacterial RNA polymerase binds its promoter on phage DNA and starts transcription
- phage DNA enters cytoplasm at 40bp/s
what are the 3 Class I genes and their roles?
- gene 0.3 –> inhibits host Type 1 Restriction Endonucleases
- gene 0.7 –> inhibits host RNA pol
- gene 1 –> T7 RNA pol
what happens when bacterial RNA polymerase binds promoter on phage DNA?
provides a motor force to pull more phage DNA into the cell
why does T7 inhibit host type 1 restriction endonucleases?
normally, these endonucleases allow bacteria to recognize and cleave phage DNA as a defense, but the phage inhibits these so that the bacteria loses its defense mechanism
why does T7 switch from host RNA pol to T7 RNA pol? how does it switch?
Class I genes are transcribed by host RNA pol, then the class I gene 0.7 inhibits host RNA pol and and gene 1 (T7 RNA pol) is activated and is more powerful and efficient to transcribe Class II and III genes
what are the roles of the 7 Class II proteins?
REPLICATION PROTEINS:
1.3 –> DNA ligase to join lagging-strand DNA
2 –> inhibit host RNA pol
3 –> endonuclease, degrades host DNA, cleaves T7 concatermer
3.5 –> lysozyme, inhibits T7 RNA pol
4 –> RNA primase/helicase
5 –> DNA pol
6 –> exonuclease, degrades host DNA
when class II proteins are being transcribed, how does the DNA entry into the cell change? why?
DNA enters at 250bp/s –> faster bc T7 RNA pol is more powerful
why do Class II proteins 3 and 6 degrade host DNA?
so the phage can use the nucleotides for itself
what are the 4 Class III proteins?
[virion structure and assembly proteins]
- portal protein
- scaffolding protein
- capsid protein
- tail protein
what proteins can host RNA pol transcribe? when does it stop transcribing?
host RNA pol can transcribe from Class I promoters but NOT Class II or III promoters –> host RNA pol is used until Class II promoters are used
describe the T7 RNA pol (2)
monomeric, 100kDa
in addition to transcribing, what is another function of T7 RNA pol that it takes over from host RNA pol
T7 RNA pol takes over from host RNA pol in transporting DNA into cell
describe T7 DNA replication (3 steps)
- T7 RNA pol makes a primer
- T7 primase/helicase deposit the primer on the 3’ end of the template
- T7 DNA pol uses RNA primer to replicate the T7 dsDNA genome (read 3’ to 5’ but making 5’ to 3’)
why will T7 DNA pol not clash with T7 RNA pol on the T7 DNA?
there is a unique ORIGIN that is 5.9kb from the left end of the genome so by the time DNA pol and primases/helicases have been made, there is no more transcription from the 5’ end anymore
why does T7 require concatemer formation? how do concatemers fix this?
DNA pol requires a primer to initiate DNA synthesis but the RNA primer cannot be removed or replaced with DNA, otherwise would lose some of the sequence
the 150bp terminal repeats pair btwn copies of phage DNA and link many copies of the genome to make a long, multi-copy phage DNA –> then 1 copy of phage DNA is packaged into each particle
is T7 DNA replication bidirectional or unidirectional?
bidirectional
is T7 DNA replication similar or different from E.coli DNA synthesis?
very similar –> also has continuous leading strand and discontinuous lagging strand
which DNA replication proteins used by T7 are from the host?
none –> T7 encodes all its own DNA replication proteins
what initiates T7 DNA replication?
T7 RNA pol
what is the family of lambda virus?
SIPHOviridae
describe the genome of lambda phage
- type of genome
- size
- unique feature
- dsDNA
- 40-60kb
- ssDNA extension (cohesive ends)
what is the purpose of lambda’s ssDNA extension/cohesive ends?
so it can pair with itself and circularize and use rolling circle replication
is lambda phage lytic or lysogenic?
TEMPERATE –> has lysogenic phase so it integrates its DNA into host DNA
what is the shape and size of the lambda head?
icosahedral head –> 63nm diameter
what is the shape and size of the lambda tail? unique characteristic
tube-like tail –> 135nm long
non-contractile
what is the receptor that lambda interacts with on the host cell?
sugar transport proteins LamB (maltose transporter)
what is a prophage?
phage has integrated its DNA into host and repressed its lytic genes so it can passively replicate with host chromosome
what is a temperate phage?
phage able to grow lytically or exist as a repressed prophage
what is a lysogen?
bacteria carrying prophage
describe the general life cycle of lambda phage
DNA enters bacteria and circularizes then either undergoes lytic growth or lysogenization
- if becomes lytic, will cause cell to lyse
- if becomes lysogenic can undergo cell division (latent) or is induced by stress to become lytic
what are the 2 types of proteins that regulate transcription initiation?
- activator
- repressor
what is the protein that regulates transcription termination? how does it work?
anti-terminator –> binds RNA pol so it continues on
describe the 2 initial transcription events after lambda infection
- host RNA polymerase uses PL and PR promoters to make phage RNA
- first 2 genes made: N and Cro
what is the role of N?
antiterminator that binds host RNA pol that is still associated with phage DNA –> lets RNA pol bypass terminators tL1 and tR2 so more phage proteins are expressed
what is another antiterminator? what is its role? is it more or less important than N?
Q
allows synthesis of downstream structural proteins from PR’ by letting RNA pol continue past tR
less important than N for controlling lytic vs lysogenic process
what 4 general types of protein does Q help produce?
replication, lysis, head, and tail proteins
what is the role of Cro?
suppresses the PRM promoter to suppress the production of CI gene
what does CI do?
suppresses PL and PR so lambda gene expression is completely shut down –> CI the master suppressor!
what happens to CI when Cro is expressed?
Cro binds PRM to block CI expression –> therefore lambda genes can be expressed
how does Cro self-regulate/
when the concentration of Cro is high enough, it can bind PR (its own promoter) and reduce its own expression
why does CI bind PR and PL while Cro binds PRM?
CI has higher affinity at PR and PL, while Cro has higher affinity at PRM
how does CI self-regulate?
when the concentration of CI is high enough, can bind PR and reduce its own expression
why does Cro need a high concentration to self-regulate?
because Cro normally has affinity at PRM, but when there’s a high enough concentration of Cro, it can bind PR to block its own production
what allows CII and CIII expression?
When N is expressed and acts as an antiterminator –> there is an increase in phage proteins
what do CII and CIII do once expressed?
CII and CIII form a dimer that acts as an activator at promoters to increase gene expression
which 3 promoters does the CII and CIII dimer act on?
- PAQ
- PRE
- Pint
what happens when CII/CIII dimer acts on PRE?
allows expression of CI
what 2 things happen when CII/CIII dimer acts on Pint?
- allows synthesis of phage integrase
- reduces expression of antiterminator Q which allows the terminator to function and reduce downstream structural proteins
describe the events when CII and CIII are active (2 series of events)
- increased CI expression –> PL and PR are blocked –> no phage genes
- integrase is expression –> can integrate phage DNA into host DNA –> prophage
how does a host cell inactivate CII?
protease
what happens to the virus if the host is healthy/metabolically active? (5 steps)
- host cell protease inactivates CII
- CII cannot activate CI expression from PRE
- PL and PR are active
- phage proteins are expressed
- phage is lytic
what happens to the virus if the host is unhealthy/not metabolically active? (7 steps)
- CII is not degraded and remains stable due to CIII expression driven by PL
- host cell proteases are inactive
- CII is in high enough concentration to activate CI expression from PRE
- PL and PR are not active
- phage proteins are not active
- integrase expression increases
- PROPHAGE (lysogenic)
is CI expressed from PRM or PRE in prophage?
CI is expressed from PRM in prophage
what does RE in PRE mean?
repression establishment
what does RM in PRM mean?
repression maintenance
in a prophage, what is the 1 gene expressed?
CI
what prevents int from being expressed normally?
Sib forms a long double-stranded stem that is recognized and degraded by RNAse, which then degrades int
what allows int to be expressed?
CII/CIII dimer activates Pint and there is no Sib present so the int gene is not degraded by RNAse
what is required for lambda DNA to integrate into host DNA to become prophage?
int and host factor
what is required for lambda DNA to be excised out of host DNA to become lytic?
int, host factor, xis
what triggers lambda DNA to be excised from host DNA?
stresses that cause DNA damage (ex. UV light)
describe the 7 steps for lambda DNA being excised from host DNA
- stress causes DNA damage
- this activates RecA protein
- RecA binds CI
- CI is cleaved and degraded
- PR and PL can be active and increase phage genes
- PL allows Xis and int to be expressed
- Xis, int, and host factor allow excision
what allows int to be easily expressed?
sib is on the opposite side of PL so PL is activated and allows xis and int to be expressed
the lambda DNA is between which 2 regions in host DNA?
attL –lambda– attR
what is an application of phage DNA integration into host DNA?
we can use as vector to clone cDNA and make cDNA library
