Transcript Powerpoint Slides 6.2 Part B

Gene Expression and
Replication in Medium DNA
Viruses
Phage Lambda
Adenovirus
What can the medium sized viruses
do that the small ones can not?
Adenovirus: more sophisticated
gene expression strategies
Lambda: two pathways for infection
Phage Lambda
Bacteriophage isolated
from human gut-Andre
Lvov?
dsDNA, 48.5 kbp
Linear, sticky ends “cos”
Complex capsid
Unusual biological
properties noticed
Model system from 1950’s
Cloning vector
The Evergreen State College Phage Lab has been a center for
undergraduate research at Evergreen since Betty Kutter came here in 1972, one
year after the college opened. Today, there are generally 10-15 students involved in
work in the lab under the direction of Kutter and faculty colleague Andrew Brabban.
We are focusing particularly on phage ecology, with the aims of understanding the
infection process under anaerobic and other conditions better reflecting those in the
natural environment, as well as on interactions between phages of different families
during simultaneous infection and the building of cocktails for potential therapeutic
applications.
Dr. Elizabeth Kutter:
[email protected]
Dr. Andrew Brabban:
[email protected]
Evergreen State-Olympia
Mansfield State (Ohio)
Bacteriophage Therapy Inst-Tbilisi
Bacteriophage therapy was used broadly in the
Soviet Union, particularly the Republic of
Georgia, which has been the global center of
phage expertise for over 80 years.
Lambda Entry and Uncoating
 Non-contractile
 Lambda
J protein
uses Lam B as
receptor
 DNA enters through
mannose permease
complex
Lambda
genetic map
Shortly after entry
the linear DNA
forms a circle
Cos sites are
complementary
with one another
Assembles
functional regions
Lambda’s two developmental
pathways
In some cases lambda follows the “usual” pathway
in which the host cell is killed straightaway-the
“lytic” pathway.
In other cases lambda DNA inserts into the
bacterial chromosome to form a “prophage”.
The prophage remains in the chromosome as a
passenger until the host cell is stressed or
“induced”.
Then it lyses the host cell to release new phage.
The latter pathway is called the “lysogenic” or
“prophage” pathway. (temperate, vegetative)
Lysis and
Lysogeny
for Phage
Lambda
What are the
advantages of
lysogeny for
lambda?
How does lambda
make its decision
for lysis or
lysogeny?
Phage lambda regulatory region
Key region regulates developmental decision
P = promoter
O = operator
T = terminator
Operon Model
Shown for lactose operon
Repressor protein binds to operator
sequence:
No mRNA so gene is off!
Inducer causes shape change
in repressor
Repressor drops off operator
mRNA produced so gene is
on
Gene Expression
Immediately after infection, N and cro are transcribed
by host RdRp (“immediate early genes”)
N is an antiterminator- it allows transcription and
expression of cIII, cII, O, Q (“early genes”).
Cro is a repressor. It can block transcription from Pr
and Pl.
N says “go”, cro says “stop”
Lytic pathway
If cro builds up quickly enough it prevents further
synthesis of N and of cro itself.
Q allows expression of “late” genes that encode for
lytic functions.
Establishment of Lysogeny
If cII builds up fast enough it activates the transcription
of cI and also some integration functions.
cI is called the lambda repressor. It shuts off all lambda
genes except itself and allows lambda DNA to
integrate into the host chromosome.
Lysis and Lysogeny Compete
If cro “wins” then late genes are expressed
and lysis follows.
If N, cII, cIII win then the lambda repressor is
expressed from cI and lysogeny follows.
This medium-sized virus is complex enough
for two developmental options.
Post Script:Lambda repressor binding
Binds to operator as a
homodimer
Helix-turn-helix protein
Up to 3 dimers can bind
Binding is cooperative
Cooperative repressor binding produces
steep response curve-”all or nothing” response
R
E
P
R
E
S
S
I
O
N
REPRESSOR CONCENTRATION
When enough repressor is around is turns off its own synthesis
When repressor levels drop-more is made
Induction involves destruction of repressor
Superinfection immunity
Lambda DNA Replication
Lambda uses a combination of host and
viral proteins.
Theta strategy for first few rounds of
replication.
Then shifts to rolling circle.
Long tail converted to ds concatemer.
Cleavage occurs at cos sites during
packaging.