Sections 1-2 Final Exam Flashcards
viruses can be grouped by:
host range, structure, genome, transmission mode
The Baltimore scheme for classification of viruses has ____ categories
Seven (7)
The Baltimore scheme categories:
- +ve ssRNA
- -ve ssRNA (goes to +ve by RdRp)
- dsRNA (goes to +ve by RdRp)
- dsDNA (goes to +ve by DdRp)
- ssDNA (goes to +ve by DP then DdRp)
- +ve ssRNA (goes through RT to make sDNA then Dp to dsDNA then DdRp)
- ss/dsDNA circle?
one of the most important and useful findings of bacteriophage research:
restriction enzymes
How were restriction enzymes discovered?
Werner Arber- Noble Prize:
a host’s range changed depending on the host strain the viral particle came from
- observed that viruses can replicate In any permissive cell but can only replicate in restrictive cells if they came from that cell lineage
- observed that upon a viruses entry into a restrictive cell, its viral DNA was fragmented (its the target)
- Restorative cells can allow a virus to be able to infect a restricted cell
2 key components to the finding of restrictive enzymes (by Werner Arber)
“r” restriction: cell has resistance to foreign DNA and produces enzyme that cuts it up
“m” modification= ability for a virus to confer resistance against “r” ad modify its DNA to protect it from the cutting enzyme
The bacteriophage piX174 controls its transcript levels through _____
- differences in promotor strengths pB>pD>pA
- Terminators stop RNA pol so transcription doesn’t always go to the end
The bacteriophage piX174 controls its protein levels through _____
- controlling transcript levels (amount of mRNA) through promoters and terminators
- Nested genes: have weak ribosomal binding sites so they are translated less often (non structural or replication)
ex. non essential, lysis, scaffolding
phage lambda discovery
Discovered by Esther Lederberg in 1951 from an E.coli strain (k-12)
in E.coli strain (K-12), phage lambda is a
lysogen (able to generate lysis) and exist as a prophage (Ur-lambda primitive)
phage lambda due to its two decision cycle it was one of the most well-studied, and they found that the structure of lambda virions ___
has an obvious pattern of gene organization verses where the proteins end up in the particles (head to tail)
a recombinant lambda version was obtained from Paris and Pasadena (Papa) and it______
did NOT have tail fibers, moves easier in soft agar, and makes larger plaques
phage lambda Papa has a ______-_____ ______ preventing its tail gene from making tail fibre proteins
therefore, not the mother of all lambda phages
frame-shift mutation
it was found that Ur-λ adsorbs to the cell better than papa-λ, due to_____
a LamB maltose transporter receptor, only recognized by the J protein on the TAIL
Which one can grow in glucose
Ur-λ (has the tail so can grow even with very little receptors
tails not essential but makes them efficient and better at binding to cells
Phage λ strains WITHOUT tails, are incapable of attaching to cells when there is ____
low amounts of receptor present
All siphoviruses have long non-contractile flexible tails of different sizes, why?
but all λs (a specific phage) have the same size
Hendrix showed that the lambda protein H is a molecular ruler to control tail length
If you delete parts of H, you get shorter tails
H size directly proportional to tail size
Similar to H, different siphoviruses have different ___ _____Proteins showing a very strong relationship between gene size and tail sizeTape Measure Proteins
Tape Measure Proteins
Phage λ DNA has 12 nt ssDNA ends called ____ _____ on the ends
Genome circularizes after entering the cell through the complimentary cos sequences
cos sites (cohesion)
Phage λ: after infection and circularization of the DNA, 2 genes are expressed: ____ and _____
those help make more genes such as ___
N and cro
cll
Phage λ: If lysis:
replication, structural and lysis genes turned on
Phage λ: If lysogeny: only 2 genes subsequently expressed – cI and int
– cI and int
DNA integrates (by protein encoded by int), then only cI is expressed during lysogeny
Phage λ: if lysis, activates expression of ____ and __ genes
structure & lysis
Phage λ: if lysogeny: expresses genes ___ and ___
cI and int
Immediately after infection, only the cro and N genes get transcribed because only the ___ and ____ are bound by the host’s RNA pol
PL and PR
transcription stops after cro and N because of the terminators
TL1 and TR1
After N and Cro get translated, they bind to DNA and RNA pol at ___ sites
nut
nut sites are named for N utilization as N is a ____ _______ protein
anti-terminator
N is an anti-terminator so it leads to continuous RNA pol transcription past terminator to make __ gene
cll
If cll is acted upon by proteases and inactivated, it means the cell is well growing and active and the _____ cycle occurs
lytic
If cll is not inactivated, It means the cell is in poor state, and the _____ cycle occurs
lysogenic
When cll gets degraded, activates replication and lysis genes and inhibits cl
cll is a transcriptional regulator. It activates expression of __ (the repressor) and __ (integrate)
cl
int
cl protein function:
turns off all the replication and lysis genes, activates its own expression
integrase
protein recombines the genome into the host genome
Integration happens by recombination between the __ (attachment phage) and the ___(attachment bacterium) sites which are homologues sequences in the two DNA molecules
attP
attB
The organization of genes integrated is different than that in the linear virion form.
Genes that were next to each other in the circular form are now at opposite ends
cl, the only gene expressed and the only protein made in the lysogenic state.
cl is a ____-_____ protein that is only able to bind DNA as a dimer (binds to diff sites w diff affinities)
DNA-binding
cl binds to 6 different sites on DNA: operators: (3) ________ which overlap promotors: (2) _____________
and operators: (3)________ which overlap promotor: (1)________
OR1,2,3 overlap PR and PRM
OL1.2.3 overlap PL
when there is ___ _____, PR and PL are left free and active so it allows for late gene expression Replication, structural, lysis
no cl
when there is __ ___ ___, all 6 sites are occupied and PRM, PR, PL are all off so no expression
a lot of cl
when there is cl, the cl proteins on the two sets of O sequences interact forming ____ _______ and makes the binding even stronger
DNA loops around
preference for binding of cl is OR1>OR2>OR3 where ____ and ____ adjacent dimers interact and have a synergistic cooperate binding effect even stronger
OR1 and OR2
The _____ is the weakest of all 6Os, last to be occupied and it overlaps PRM “Promotor for Repressor Maintenance”
OR3
if cl levels decrease OR3 is the first to be vacant, frees up ____ and activates cl transcription
PRM
This cells cell in lysogenic state
cells carrying lambda integrated as a prophage are protected from super-infection by other lambda particles (and similar things) as there is a lot of ___ protein that can prevent expression of all incoming phage genes
cl
lambda (and other prophages) can be included to enter lytic cycle by _____ of the cl protien from its DNA and prevention of its binding
HOW??
REMOVAL OF cl protein TO ENTER LYTIC
induction of the lytic cycle occurs when the cl protein is degraded by the bacterial protease ____
RecA
cleaves and inactivates cl preventing its binding to DNA
how does RecA work: (2 things)
- happens in response to hosts SOS response (bacteria response to stress such as DNA damaging agents)
- cleaves and inactivates cl preventing its binding to DNA
RecA is normally a _______ _____ but in response to DNA damage it becomes active as a protease
recombination enzyme
RecA targets cellular repressors and removes them especially ______ which triggers the SOS response
LexA
LexA normally inhibits expression of _____ _____ so when its removed all the DNA damage stress genes get expressed
stress genes
LexA and cl look alike so RecA recognizes it by mistake and it is a way the phage co-opted the cell’s damage control system as a cue for activating its lytic replication AKA:
things are going bad for this cell lets get out
removal of cl repressor exposes PR and allows for transcription of cro protein
Right promotor
Cro turns on expression of early lytic genes and inhibits ___ transcription
cl
upon induction ______ occurs, where the phages genome comes out of its integrated into the cells chromosome state into a circle
excision
integration requires ____ and excision requires __ and ____ both are translated from the mRNA at the end of the integrated genome
int
int and xis (excisionsase)
The genome must be in circular excised from for the lytic pathway to :(2 things)
- replicate the DNA
- express late genes
During the lytic cycle: Integration into the host genome is prevented by a phenomenon called _____
retroregulation
Retroregulation: anti termination by N allows PL transcription to proceed past int and xis to ____
sib
sib does not encode a protein, so what does it do?
has a sequence that gets targeted by the cellular RNase enzymes, which degrade the mRNA in a 3’ to 5’ direction
due to sib, more xis gets translated before the RNase makes it to it as compared to int, this disproportionate ratio keeps lambda DNA out of the chromosome ONLY WHEN
its in the excised circular form
when its inside the chromosome to begin with, sib isn’t downstream of int cuz of the attp, so int and xis get transcribed to allow excision, then sib prevents int translation so that the genome can’t reintegrate
why must the genome be In circular form for the lytic cycle?
- cuz the structure gene promoter is at the other end of the linear version of the genome that is inside visions
- cuz the mechanism for DNA replication for phage lambda is the rolling circle replication of a long concatemer that joins at cos sites
SaPIs are
Staphylococcus aureus pathogenicity islands
diff SaPIs are present in diff Staphylococcus aureus strains to encode various _____ factors that contribute to bacterial pathogenicity
virulence
SaPIs are normally not active but they are induced for replication by ______ _____ that have a protein that induces SaPIs to make a protein that hijacks the phage capsid protein and packaging machinery (makes phages with shrunken heads and SaPI DNA)
replicating phages
lysed cell releases SaPIs ____ _____and some of the original helper phage ____ _____
smaller heads
larger heads
released SaPI can
1- go and infect other cells
2- Integrate SaPI DNA into cells genome (recombination at att sites like lambda)
3- SaPI inactive for replication until helper gene comes
Results in transfer of virulence factors from cell to cell
SaPI encode repressor protein that keeps them inactive ____ (acts as sensor to see if helper is there)
when helper phage comes, it encodes the antirepressor protien and SaPI is activated
Stl
SaPI proteins inhibit
helper Phage gene replication
shrink the head
alter the terminase packaging enzymes so that SaPI genes get packaged
SaPIs were the first to be discovered, now there is a lot of PICIs “___”
phage-inducible chromosomal islands
CRISPR stands for
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPER was identified in bacterial genome sequences and it was shown that different isolates of the same species have different
and that they are always associated with a set of genes:
sets of spacers
cas: crisper assosicated
They then noticed that those CRISPER spacers were similar to sequences found in:
plasmids and phages
____________ ___________ is an important bacterium used in the production of yogurt and cheese
Streptococcus thermophilus (associated CRISPER by Canadian researcher in U LAVAL)
took Streptococcus thermophilus and exposed it to phages and isolated cells that survived the infection, found out:
1- these cells are now more resistant to infection by one or both phages (immunity)
2-the resistant strains had additional new spacers in their CRSIPR region at the left end of the array and they matched sequences from phage genomes
(less matches meant less resistance)
3- mutant phages were found with changes in the region corresponding to the resistance spacer, can replicate in cells resistant to the wild type
New spacers in resistant LAB cells matched sequences from phage genomes, but partial match = partial (less) ____
resistance
phages can bypass CRSIPR resistance by:
- phages can become mutated (in regions that correspond to spacer making them unrecognizable)
- phages can produced proteins that inhibit CRISPR-Cas systems
some cas proteins:
some cas proteins:
-acquiring the new spacers
-carry out subsequent gene silencing task
steps of CRISPR cas model of function
- the repeat spacer region in transcribed into RNA
- repeats fold into hairpins due to palindrome sequences
- cas proteins process these repeats and cut them into small spacer containing RNAs (sRNA)
- take the sRNA to target the matching phage or plasmid DNA
V.C has SAPI-like island to protect it from phage BUT
phage (new!) has Crisper cas, with spacers that match SAPI
SO:
Absence of phage CRISPER cas: no viral replication as its blocked by SAPI-like islands
Presence of CIRSPER-cas but no SAPI-like spacers, greatly reduced viral replication but some phages pick up the spacer and gain resistance
Presence of CIRSPER-cas with SAPI-like spacers: good viral replication and protection from SAPI degredation
