Lecture 18- Bacteriophage Lambda II Flashcards
Different states of viral genes in latent states for temperate viruses
Integrated into cellular DNA or persist as plasmids.
Virus in latent state; viral genes must persist somehow. There are two fundamental ways.
Integrated into cellular DNA
Phage lambda, SV40, adenoviruses, retroviruses
Physically integrated.
Need to maintain the viral genes. Need guarantee that viral genes will be passed on to progeny cells. This is a sure way for viral DNA to persist in the latent stage.
Persist as plasmids
Phage P1, herpesviruses, bovine papilloma virus
Much less fool proof. DNA does not integrate, but stays as a plasmid. There are thousands of plasmids, cell just cuts in half. Chances are good that both daughter cells will receive some of the plasmid. Some of these have a relatively small number of plasmid molecules in the cell. There is the chance that one cell would get all plasmids and the other would get none.
Bovine papilloma virus only has a few plasmids BUT has a partition mechanism that ensures that each cell will get at least one plasmid.
Circularity of lambda genome
The lambda genome is functionally circular (linear in virion but functions as a circle)
1) Replication (theta and rolling circle replication) (rolling circle later in replication)
2) Late gene expression (structural genes expressed at late times after infection; promoter for late genes expressed at early times)
3) Integration (site specific reciprocal recombination)
Why is the genome linear in the virion?
Maybe:
Injection of the genome into the bacteria easier if linear
also, if linear, might be linear b/c that’s the form that’s easiest to put through the portal vertex during assembly
Bacteriophage lambda life cycle
For 10 min, a lot happens but decision is not made on which route to take.
***Site specific recombination of integration requires inserted or integrated prophage- how?
During non productive response: integration, get a prophage. Upon induction of lysogen: excision. Excision is also site specific
Lambda integration
Genome: A J att lambda N R
att lambda flanked by p on left and p’ on right
circularizes so A and R are on top, p and p’ and att lambda on bottom
Key: enzymatic activity responsible for entire reaction is found in the integrase protein (lambda int)
IHF only plays a structural role; no enzymatic activity
att L and att R look like att B and att Lambda
low frequency, a second lambda circle can integrate into the dna. it can do this by having an interaction between att lambda and att L or R. then you get a double lysogen.
things needed to integrate: lambda int, lambda xis protein, IHF +FIS, lambda int protein, E coli IHF (integration host factor, 2 proteins)
p = phage sequences to the left and right of the att site
att lambda is the attachment site on Lambda DNA
substrate for integration reactions is circularized dna
att lambda has 3 sites for IHF and 7 sites for Int
complex when it assembles at att is called an intosome
2 integrase proteins have binding sites exposed and capture att b to initiate reaction (att b is bacterial att site)
att b 10x shorter than att lambda site- significance?
b with p’ at att L and p with b’ at att R
remember that integrate and IHF are involved in the integration reaction
integration reaction: p to b’ and b to p’
Lambda integration vs excision
After infection- need int protein
After induction of a lysogen- need both int and xis (need both)
For excision, need IHF and a second host factor FIS
Second lambda protein: lambda excise protein (Xis) also required
no enzymatic activity for xis
xis binds to att R (not att L)
Mechanism of site specific recombination
crosses over, and p binds to b’ and p’ binds to b
all att sites contain the same core sequence of 15 bp
int recognizes this sequence and makes a 7 bp staggered break in the dna (staggered 7 bp cleavage)
cleavage reaction does not involve just simple hydrolysis of a phosphodiester bond
always covalently bound to 3’ end at the site of cleavage
just like topoisomerase!
advantage of covalent bond: no energy loss when religating
religation by int
functions like topoisomerase
Transduction
Definition: the transfer of cellular genes from one cell to another by virus factor
key reminder: all enzymology done by lambda integrase (double check)
Generalized transduction:
Transduction in which a set of host genes are acquired at random and transferred to another cell
Ex: bacteriophage P1, headful packaging
when host genes are acquired at random and any host genes can be picked up. no specificity to process
mostly incorporates host dan instead of phage dna
T4 uses headful mechanism, but doesn’t carry out gen transduction b/c it degrades host dna very early in infection
Specialized transduction:
transduction in which only a very limited group of cellular genes can be transferred to another cell by a virus
ex: bacteriophage lambda, gal and biotin genes
only a small subset of total genes in a cell are transferred to another cell by the virus
carried out very efficiently for gal and biotin
must have something to do with the genes that flank att b
Mechanism of specialized transduction
non homologous recombination
ex: cuts from gal to right before J instead of att L to att R
does not include J because that would make it too large. Lambda has a packaging limit in head
1 in 100000 induced cells have a non homologous recombination event
Means no sequence specificity. Almost at random w/ any two sites in the DNA.
Transduced genes adjacent to phage att site in host genome
in this example, will generate this circular dna which will contain galactose genes linked to phage genes
how would you detect this rare event? have cell you infect to be gal -
made mutations in one of the genes in gal operon
mutant gene could be repaired
get a lambda dg (defective gal transducing particle)
why are all gal transducing particles missing lambda genes?
missing the J gene- codes for the tail protein
get particles produced, some with wild type phage lambda, some with dg lambda
non specific- illegitimate recombination
lambda biotin genes not usually defective- double check this
