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BC21C: Molecular Biology 1BC21C: Molecular Biology 1
DrDrSherlineSherline BrownBrown
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BacteriophageBacteriophage
Learning ObjectivesLearning ObjectivesTo explain gene organization and expression inTo explain gene organization and expression in
bacteriophagebacteriophage
To describe the role of CI andTo describe the role of CI and CroCro in transcriptionin transcription
regulationregulation
To explain the factors determiningTo explain the factors determining lyticlytic growth andgrowth and
lysogeniclysogenic growthgrowth
To describe antiTo describe anti--termination andtermination and retroregulationretroregulation
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BacteriophageBacteriophage
Course OutlineCourse OutlineMorphology ofMorphology ofbacteriophagebacteriophage
Gene organization inGene organization in bacteriophagebacteriophage
Gene expression inGene expression in bacteriophagebacteriophage
BacteriophageBacteriophage gene functiongene function
TheThe immnunityimmnunity regionregionControl of transcription by CI andControl of transcription by CI and CroCro
Factors determiningFactors determining lysogeniclysogenic andand lyticlytic growthgrowth
LysogenicLysogenic growth ofgrowth ofbacteriophagebacteriophage
LyticLytic growth ofgrowth ofbacteriophagebacteriophage
AntiterminationAntitermination andand RetroregulationRetroregulation
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Biology & GeneticsBiology & Genetics
ofof
BacteriophageBacteriophage
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Bacteriophage
What is a bacteriophage?
A bacteriophage is a
virus that infects
prokaryotic cells.
Affect enteric bacteria
such as E. coli andSalmonella typhimurium
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Bacteriophage
Why study
bacteriophages ?
Viruses represent the
ultimate in parasites
Unique system for
studying regulation ofgene expression as well
as protein - protein
interaction
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Bacteriophage
Bacteriophages arediverse
Can have dsRNA or ssRNA genome
Can have ss DNA ordsDNA genome
Best studied are thosewith dsDNA genome
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Bacteriophage
A phage can either be
virulent ortemperate
Virulent phages: lyse or
kill their hosts after
infection (lytic)
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Bacteriophage
Tempererate phages: can achieve a state
where their genome
replicates along with the
host genome without
killing it (lysogenic)
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Bacteriophage
Lytic growth:
you have replication of
phage DNA and
synthesis of new coat
proteins. They combine
to form new page
particles that are released
by lysis of host cell
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Bacteriophage
Lysogenic growth:
Involves integration of
phage DNA into the
chromosome of the host
cell where it replicates
during cell division
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Bacteriophage
Prophage: integrated
phage
Lysogen: bacterium
harbouring the prophage
Lysogenic bacteria has
immunity against furtherinfection from a similar
bacteriophage
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Bacteriophage
Induction: switch fromlysogenic to lytic pathway
Lysogeny is ofecological importance
Best studied temperatephage is lambda
Lambda has both lyticand lysogenic pathway
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BacteriophageBacteriophage
MorphologyMorphology
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Bacteriophage
Morphology
Lambda virion is similarto that of other tailed
bacteriophages however
no tail fibers are present
Genome consists of a
linear ds DNA molecule
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Bacteriophage
Morphology
Genome consists of a linear ds
DNA molecule
Size 48, 502 base pairs
The 5` terminus of each strand
has a single tail (12 nt long)
These single stranded ends
are complementary (ends are
said to be cohesive)
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Bacteriophage
When the two ends of the DNA are free in the host cell
they associate (the cos site)
DNA is ligated to form a double stranded circle
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Bacteriophage
Best studied host- virussystem
Led to increase inknowledge about phage
and their development
Uncovered unknown
information about hostfunction
Recombination, generegulation, protein folding
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BacteriophageBacteriophage
Gene organizationGene organization
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Bacteriophage
Gene organization
Lamda genome divided into functional units
Morphogenesis
Recombination
Regulation
Replication
Lysis
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Bacteriophage
Gene organization
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Bacteriophage
Gene organizationHead & Tail
Genes code for structuralprotein of bacteriophage
capsid
Codes for terminase enzymes
required to process rolling
circle multimers into unit
genome-length pieces during
packaging
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Bacteriophage
Gene organizationRecombination
Code for Int and Xis geneswhich are required for integration
of the bacteriophage into the
bacterial host chromosome
Codes for excision of
bacteriophage from the bacterial
host chromosome during
induction
Codes for other genes as well
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Bacteriophage
Gene organizationRegulation
Includes the immunity region as
well as genes that are
responsible for the genetic
switch
The Q antiterminator protein aswell as the anti-Q RNA and PR`
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Bacteriophage
Gene organization
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Bacteriophage
Gene organizationReplication
This region includes 2
replication protein genes O
and P and the origin of
replication
B i h
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Bacteriophage
Gene organizationLysis
There are 4 genes in the lysis
region
B t i h
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Bacteriophage
Gene organization
Some gene products and their function
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BacteriophageBacteriophage
Gene ExpressionGene Expression
B t i h
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Bacteriophage
Gene Expression Bacteriophage gene
expression can be classified
into 3 phases:
Very early expression
Early expression
Late lytic or lysogenic
expression
B t i h
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Bacteriophage
Gene Expression
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Bacteriophage
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Bacteriophage
Gene Expression PL expresses the anti-
terminator protein N
PR expresses the anti-
repressor protein cro
PR` pauses after a short
distance and no protein is
expressed
Bacteriophage
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Bacteriophage
Gene Expression Early expression depends
on the action of the N
protein
The N anti-terminator
causes expression fromPR and PL to continue
past transcription
terminators
Bacteriophage
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Bacteriophage
Gene Expression
CII & CIII favour
lysogenic growth
O & P are required for
replication
Q favours the lytic growth
Bacteriophage
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Bacteriophage
Gene Expression Only genes expressed
are the lysis genes and
the genes coding forhead and tail proteins
The anti-terminatorprotein Q is required for
expression of these
genes
Bacteriophage
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Bacteriophage
Gene Expression
At the same time, cro will
Prevent any further gene
expression from PR orPL
Bacteriophage
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Bacteriophage
Gene Expression
The only genes
expressed are int and cI
Once the bacteriophage
has integrated into the
bacterial host
chromosome, cI is the
only gene that willcontinue to be expressed
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BacteriophageBacteriophage
The Immunity RegionThe Immunity Region
Bacteriophage
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Bacteriophage
The Immunity Region
The immunity region of lambda contains:
Three Promoters: PL, PRM, and PR`
Two Operators: OL and OR
Two Genes : c1 and cro
OR3 OR2 OR1
PRM PR
OR
OR3 OR2 OR1
Bacteriophage
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Bacteriophage
The Immunity Region
The immunity region of lambda contains:
Three Promoters: PL, PRM, and PR`
Two Operators: OL and OR
Two Genes : c1 and cro
Bacteriophage
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Bacteriophage
The Immunity Region
The Operators
The operator is a specifc
region of the DNA at the
initial end of a gene where
the repressor protein
binds and blocks mRNA
synthesis
OL and OR consists ofthree operator sites to
which either repressorcI
orcro can bind
OL1
OR1
OL2
OL3
OR2
OR3
Bacteriophage
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Bacteriophage
The Immunity Region
The Operators
Each operator site is 17
bp long and has a center
of dyad symmetry
Each operator half site
binds with one monomer
of repressor orcro
OL1
OR1
OL2
OL3
OR2
OR3
Bacteriophage
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acte op age
The Immunity Region
The Operators
Repressors binds to each of the operator
sites with different binding affinities
OL1 > OL2 ~ OL3
OR1 > OR2 ~ OR3
Bacteriophage
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p g
The Immunity Region
The Operators
CRO binds to each of the operator sites with
different binding affinities
OL3 > OL2 ~ OL1
OR3 > OR2 ~ OR1
Bacteriophage
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p g
The Immunity Region
The operator-sites are not
identical in sequence
The OR3 operator-site is the
only one with a T:A base pair
at position 3 in both halves of
the operator-sites.
This is the site to which crobinds with the highest affinity
OL1
OR1
OL2
OL3
OR2
OR3
Bacteriophage
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p g
The Immunity Region
The Promoters
A promoter region is the site on
the DNA where RNA
ploymerase binds and begins
transcription
OL contains a single promoter
PL
Bacteriophage
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p g
The Immunity Region
The Promoters
PL directs transcription in a
leftward direction through the N
gene and eventually through
the recombination region
PL is a strong promoter
Bacteriophage
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The Immunity Region
The Promoters
OR contains two
promoters PR and PRM
PR directs transcription in
a rightward directionthrough cro and
eventually through the
replication region
PR is a strong promoter
Bacteriophage
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The Immunity Region
The Promoters
PRM directs transcription
in a leftward direction
through cI
PRM is a weak promoter
Bacteriophage
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The Immunity Region
PRM overlaps OR2 by 2 base pairs
PR overlaps OR2 by 3 base pairs
Difference allows repressor bound to OR2 to activate transcription
from PRM but repress transcription from PR How are these promoters controlled? This is where the repressor comes in
Bacteriophage
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The Immunity Region
Repressors
The repressor protein is a
regulatory protein that binds to
specific sites on DNA and
blocks transcription, it is
involved in negative control
Bacteriophage
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The Immunity Region
Repressors
Small polypeptide 27 kD
N-terminal domain is the
operator binding site
N- terminal has a helix-turn-helix (HTH) motif
C-terminal domain responsiblefor dimerization
Bacteriophage
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The Immunity Region
Repressors
2 domains joined by aconnector
Each of the operator sitesin OR and OL binds arepressor dimer
Each individual Nterminal region contacts ahalf site
Bacteriophage
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The Immunity Region
Repressors
The repressor is a H-T-H
helix binding protein
The 2 alpha helices of the
HTH motif in adjacent
repressor monomers
contact each half of anoperator site
Bacteriophage
Th I it R i
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The Immunity Region
Repressors
The amino acid sequence
of the recognition helix
makes contact with
particular bases in theoperator sequence that it
recognises
Bacteriophage
Th I it R i
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The Immunity Region
Repressors
Cleavage of the domain
can occur with the use of
papain or the recA
protein.
This results in induction
Bacteriophage
Th I it R i
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The Immunity Region
Repressors Repressor binding to one
operator increases theaffinity for binding of arepressor dimer to theadjacent operator
The affinity is 10 Xgreater forOL1 and OR1than for the otheroperators so they bindfirst
Bacteriophage
Th I it R i
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The Immunity Region
Repressors Co-orperativity allows
the repressor to bind the
OR1 and OR2 sites at a
lower concentration
Bacteriophage
The Immunity Region
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The Immunity Region
Repressors Coorperative binding increase
the effective affinity of therepressor for the operator at
physiological concentration
Allows a lower concentration of
repressor to achieve
occupancy of the operator
Why does this have serious
consequences?
Bacteriophage
The Immunity Region
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The Immunity Region
Repressors
Repressor binding shows coorperativity
OR1 ~ OR2 > OR3
Bacteriophage
The Immunity Region
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The Immunity Region
The Cro Protein
Very small protein
66 amino acids
Consists of a single domain
which contains a helix-turn-
helix DNA binding motif
Bacteriophage
The Immunity Region
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The Immunity Region
The Cro Protein
Order of binding affinity is
different
OR3> OR2 ~ OR1
Bacteriophage
The Immunity Region
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The Immunity Region
The Cro Protein Binding of the Cro protein to the
operator site is similar to that of the
repressorexcept
Cro does not bindcooperatively
Cro does not have aminoterminal arms
Cro forms a specific bond withT:A base pair at position 3 ofthe operator half site. Thissite is not recognized bylambda repressor
Bacteriophage
The Immunity Region
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The Immunity Region
Lambda repressor acts as a transcription
activator whereas cro is unable to do so
Bacteriophage Control of transcription at O by the repressor
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Control of transcription at OR
by the repressor
OR contains 2 promoters: PR and PRM
RNA polymerase will readily bind to PR and initiate transcription
in a rightward direction through Cro
RNA polymerase may under the right conditions bind to PRM
and initiate leftward transcription through the CI gene
The ability of RNA polymerase to bind to PR and PRM and initiate
transcription depends on the conc ofCro and CI
Bacteriophage
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Repressor bound at
OR2 blocks expression at PRbut activates PRM. This is due
to the fact that:
PR overlaps OR2 by 3 bpwhile PRM overlaps OR2 by
3 bp
Repressor can formcontact with RNApolymerase that increasesits binding affinity for apromoter
Acts as a transcription activator
Bacteriophage
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Repressor bound at OR3blocks expression at PRm.
Bacteriophage
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BacteriophageBacteriophage
LysogenyLysogeny
Bacteriophage
Lysogeny
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Lysogeny
Establishment of lysogeny CII is transcribed by PR and CIII is transcribed by PL
CII binds upstream ofPRE and stimulate the transcriptionofCI from PRE (CII can be described as?)
Only when sufficient repressor has been made by PRE thatit can bind to OR1/OR2 and direct its own synthesis
Bacteriophage
Lysogeny
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Lysogeny
Establishment of lysogeny
CI also activates PRM and directs more of its own synthesis
CII also activates PI
PI allows the transcription of the Int gene
The Int enzyme integrates the lambda DNA into the bacterial DNA
Bacteriophage
Lysogeny
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Lysogeny
Establishment of lysogeny
CII also activates PaQ
Transcription from PaQ makes a short selfterminating transcript that
function in antisense control ofQ expression
Transcription from PRE and PaQ serves to counteract the effects ofcroand Q which promote lytic growth
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Bacteriophage Lysogeny
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Establishment of lysogeny
CIII inhibits cellular proteases that degrade CII
IfCIII is absent CII will be rapidly degraded and no lysogen will be
formed
Bacteriophage
Lysogeny
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y g y
Establishment of lysogeny The level of CII in the cell is dependent on two proteases
HflA and HflB (FtsH)
The level of the host proteases is dependent on the growth state of thebacteria
Well fed E. coli is high in proteases
Starving E. coli are low in proteases
Bacteriophage
Lysogeny
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y g y
Establishment of lysogeny
The level ofCII in the cell depends on: The gene dosage ofCII and CIII in the cell
If lambda infects E.coli at a multiplicity of infection (moi)of 1 phage /cell, insufficient CII will be expressed toactivate transcription and lysogenic growth
Ifmoi is 10 phage/cell the amount ofCII and CIII willincrease
The extra CIII will protect CII
Bacteriophage
Lysogeny
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y g y
Establishment of lysogeny
The dependence of lysogeny on moi in the cells explains
why bacteriophage lambda forms turbid plaques
The dependence of lysogeny on moi also explains why
induction is irreversible
Bacteriophage Integration of
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Bacteriophage lambda
circularizes after infection
Int promotes
recombination between
the attachment sequence
(attP) on the phage
protein and a site on thebacterial DNA (attB)
Bacteriophage Integration of
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The recombinationpromoted by Int is sitespecific
This site specificrecombination is
nonhomologous, only ashort region of homologyis present (7 bp)
In light of this Int isneeded because itrecognizes attP and attB
Bacteriophage Maintenance of
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After the lysogen has been formed the CI repressor is
one of the few genes to be transcribed
In the prophage state the CI gene is transcribed by PRMinstead ofPRE
Bacteriophage Maintenance of
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Regulation of repressor synthesis in the lysogenic state
CI repressor is functional only as a dimer
At very low concentrations ofCI polypeptides dimers cannot be
formed
At optimal concentration CI repressor binds to OR1 then OR2.
If conc is too high it will bind to OR3 and regulate its own synthesis
Regulation of repressor synthesis in the lysogenic
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state
Bacteriophage Maintenance of
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Immunity to superinfection
The CI gene regulates the transcription of any otherlambda phage infecting the lysogenic cell by binding to
the operators of the phage
Hence immunity to lambda super infection
However similar phages with a different operator
sequence can infect the lysogen
Bacteriophage Induction
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will remain a prophage until
the cell is damaged
The RecA when complexed
with ssDNA can activate the
protease activites of other
proteins of the host.
Brings about the autoclevage
of LexA protein
.
repressor is similar to LexA.
Proteolytic cleavage of the CI
repressor also accurs
Bacteriophage Induction
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Dimerizationis lost
Repressor drops offoperators
Transcription initiatedfrom PL and PR
Lytic cyle
Bacteriophage Induction
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Role of cro protein in induction
Cro gene product is one of the first proteins to be produced afterinduction
Cro prevents the synthesis of more repressor
Cro binds first to OR3 then OR2 preventing the repressor from binding
to OR2
Cro binds to OL3 prevents repressor binding to OL
PL is no longer repressed
Bacteriophage
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Role of cro protein in induction
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Bacteriophage Exision
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Transcription from PL and PR can now take place
PL transcribes Int and xis
Exision requires both genes because the recombination
occurs between different sequences from those used forintegration
Integration required combination between attB and attP
Bacteriophage Exision
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Exision requires recombination between the hybrid
attP-attB sequences that exist at the junction between the prophage
DNA and the chromosomal DNA
This sequence is different from those at attP orattB
Int alone cannot exise the DNA, it can only take place when xis ispresent
The molecular basis for this is described as retroregulation
O and P genes are transcribed by PR (what process is this required for)
Bacteriophage Exision
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Bacteriophage Lytic growth
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Very early expression
When bacteriophage has infected a cell and injected its DNA
genome will be transcribed by host RNA polymerase from 3 strong
promoters:
PL, PR and PR`
The first genes to be expressed after bacteriophage lambda infectionare N and cro
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Bacteriophage Lytic growth
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Early Expression
The early transcript ofPR codes for
Cro anti-repressor
CII transcription activator required for lysogenic growth
O& P required for replication of the bacteriophage
Q anti-terminator protein required for late geneexpression
Bacteriophage Lytic growth
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Early Expression
The early transcript ofPL codes for
CIII required to protect CII
Xis required for exision of the prophage
Int required for integration of the prophage
Transcription continues beyond the sib site
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Bacteriophage Lytic growth
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Early expression
sib is an RNase II processing site
Any transcript which passes through sib will form a hairpin structure
that will be cleaved by RNase III
The free 3` ends will be degraded by exonuclease.
Int and Xis will be degraded before it can be translated
This type of regulation is called retroregulation
Bacteriophage Lytic growth
L t E i
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Late Expresssion
Late gene expression depends on the expression ofQ and cro
proteins
Q allows PR` to transcribe the lysis genes (S, R, Rz) and the capsidproteins (Nu1, A, W etc)
Cro binds to the 3 operator sites in OL and OR
Cro represses all further transcription from PL and PR
Prevents further expression ofN, CII, CIII so lysogenic growthcannot occur
Bacteriophage
Steps leading to lytic and lysogenic development
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Steps leading to lytic and lysogenic development
Bacteriophage
Steps leading to lytic and lysogenic development
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p g y y g p
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BacteriophageBacteriophage
TranscriptionalTranscriptional AntiterminationAntitermination InIn
Bacteriophage
Antitermination
Th t tit i t N d Q
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The two antiterminators are N and Q
uses antitermination regulation at two stages of itsdevelopment
Early Stage (regulates synthesis of recombination and replication
functions)
Late stage (regulates synthesis of late genes including head andtail proteins)
Bacteriophage The N protein
When DNA infects a cell 2 rRNA are transcribed that synthesizes
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When DNA infects a cell 2 rRNA are transcribed that synthesizesthe N and Cro genes. Then it stops
Initial transcription initiated at PR promoter terminates at TR`
One of the sequences transcribed into RNA is nutR (N utilizationrightward)
Bacteriophage The N protein
N will bind to RNA polymerase after the nutR region has been
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N will bind to RNA polymerase after the nutR region has been
transcribed.
RNA polymerase with N added will transcribe past the TR` site
Similar events occur on the left side
Bacteriophage The N protein
The protein binds to the nut site sequence on the mRNA rather than
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The protein binds to the nut site sequence on the mRNA rather than
on the DNA
The nut sites consists of a sequence called BoxB that N binds to
N changes its conformation after binding to BoxB
In this state it is able to bind to RNA polymerase and prevent
termination
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Bacteriophage The N protein
The host proteins collaborate with N in causing anti-
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The host proteins collaborate with N in causing anti-
termination
These are called nus protein
Nus proteins are (nusA, nusB, nusE, nusG)
Bacteriophage The Q protein
Q is a gene under the influence of the N anti-terminator
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Q is a gene under the influence of the N anti terminator
Q allows transcription from the late promoterPR` toproceed through terminators into downstream genes
Q loads on to RNA polymerase in response to asequence (QBE orqut) located close the promoter
Bacteriophage The Q protein
The qut sequence is not found totally in the mRNA,
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The qut sequence is not found totally in the mRNA,some of the required sequence is in the DNA (nottranscribed)
In the absence ofQ, the polymerase binds to PR` andinitiate transcription
It pauses after 16-17 bases have been transcribed andterminates at TR`
Bacteriophage The Q protein
In the presence of Q Q binds to qut once the
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In the presence of Q, Q binds to qut once thepolymerase has left the promoter and transfers to the
paused polymerase, allowing it to transcribe through TR`
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Bacteriophage Retroregulation
The int mRNA initiated at PL is degraded by cellular
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L g y
nucleases
The int mRNA initiated at PI is stable and can be
translated into integrase protein
RNA initiated at PI stops at a terminator 300nt after the
end of the int gene.
It forms a stem loop with 6 uridine bases
Bacteriophage Retroregulation
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Bacteriophage Retroregulation
When RNA synthesis initiated at PL, the RNA
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y Lpolymerase is modified by N and goes beyond the
terminator at the end ofint gene
This longer mRNA forms a stem that is the substrate fornucleases, hence it is degraded .
Bacteriophage Retroregulation
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Bacteriophage Retroregulation
When the phage genome is integrated in to the host
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chromosome the site causing the degradation is
removed from the end of the int gene
Int mRNA can be made from PL