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BMB400, 4_4_04
BACTERIOPHAGE
Regulation of Gene Expression in
Bacteriophage
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• Phage – A virus, it is one kind of vector.
• Bacteriophage – a phage (virus) that infects bacteria
• Simple “organism”
– Basic structure consists of DNA or RNA surrounded
by a protein coat
– Vary greatly in shape and size
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Visible effects
on DNA during
viral infection
T4 phage
DNA
Pre-infection
Post-infection5
• Phage cannot replicate on
their own
– Infect a host cell
• Once inside the host cell,
they either remain
quiescent (as prophage)
or use the cell’s
replication machinery to
produce many copies of
themselves
– This replication may or
may not lead to
destruction of the host
cell (lytic)
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Bacteriophage
• Why are phage important?
– They cause diseases
– They are useful tools in molecular biology
• Phage are often used as “replacement vectors”
- Part of their DNA is removed and replaced with other DNA
of interest (e.g., human DNA)
- This recombinant DNA is repackaged into phage; phage
infect host bacteria
- Phage use bacteria to replicate, therefore replicating
the DNA of interest
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 bacteriophage
as a replacement
vector
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Phage conversion
Dormant prophage – integrated bacteriophage – carries
genes that alter the phenotype of the microbe
- best examples are pathogens and toxin production
toxin
prophage
Corynebacterium
diptheriaea
Phage produces
diptheria toxin
This is what makes
people sick
insertion
site
C.diptheriaea
without phage strain
produces no toxin
Does not cause
diptheria
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Lysis or Lysogeny
• Lysis: Infection by phage produces many
progeny and breaks open (lyses) the host
bacterium (bacteriophage T4)
• Lysogeny: After infection, the phage DNA
integrates into the host genome and resides
there passively
– No progeny
– No lysis of the host
• Bacteriophage lambda can do either.
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The lytic pathway of bacteriophage infection
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Phage life cycle:
Lytic vs. Lysogenic pathways
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Elements of lysogeny
• The phage genome integrated into the host
bacterial genome is a prophage.
• Bacterium carrying the prophage is a
lysogen.
• Lysogens are immune to further infection by
similar phage because the phage functions
are repressed in trans.
• Induction of the lysogen leads to excision
of the prophage, replication of the phage
DNA, and lysis of the host bacterium.
Site-specific recombination in
bacteriophage .
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X-Ray structure of integration host
factor (IHF) in complex with a 35-bp
target DNA.
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The Bacteriophage T4
• Early translation
– products are usually enzymes to shut down host
and synthesize viral nucleic acids
• Late transcription/translation produces structural
proteins
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Lytic virus
T4 has linear DNA with specific hydroxymethylcytosine, instead of
cytosine, resistent to endonuclease of the host
Produce enzyme in DNA replication that similar to those host
enzyme in large amount
Encode three group protein (early, middle and late proteins)
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A genetic map of bacteriophage .
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The Bacteriophage lambdha
Lytic and lysogenic virus
Genome: double strand DNA, has cohesive ends (cos)
Early transcription begin on PL and PR to give the product of N
and Cro protein (regulator)
N antiterminator (produce O, P (induce replication), Cll and Clll
The antiterminator is not effective before termination before the Q
gene (little Q protein is produced, Q is antiterminator to transcribe
the late genes))
Cro regulator between lytic dand lysogenic pathways by blocking
PL and PR (binding OL and OR (site 1, 2, 3)
CII (aktivator protein by activate PE to express CI and integrase)
and CIII is stabilization of CII.
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lytic pathway
lysogeny pathway
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Lytic and lysogeny pathways
Transcription is regulated by
• Repression (initiation)
• Activation (initiation)
• Anti-termination (elongation)
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Repressor structure
 repressor (CI) is a dimer; monomer has 236 amino acids.
C-terminal domain: protein-protein interaction;
dimerization and cooperativity
Connector
N-terminus: DNA binding; Helix-Turn-Helix motif
operator
repressor can bind cooperatively
to operator sub-sites.
operator
oR2
operator
oR1
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Cro structure
Cro is a dimer
Monomer has 66 amino acids
Has only one protein domain
Does NOT display cooperativity
DNA binding; Helix-Turn-Helix motif; also dimerization
operator
oR3
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Page 1409
Voet Biochemistry 3e
© 2004 John Wiley & Sons, Inc.
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Theoretical repression curves for  pR (right) and a simple
repressor–operator system such as that of the lac operon
(left).
Bacteriophage : Events leading to lysis
• lysis or lysogeny (cI or Cro?) ?
• Both lysis and lysogeny:
– PR, PL, active : synthesize N, Cro
– antitermination by N : synthesize cIII, cII, Q
• Lysis:
– Low [Cro] : binds OR3, shuts off PRM (cI)
– High [Cro] : shuts off PR and PL
– antitermination by N protein to produce Q
Protein which activate late genes
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Factors favoring lysogeny
• High multiplicity of infection
– More templates produce more of the CII protein, which
stimulates PRE.
– Phage sense that it is too crowded.
• Poor nutrient conditions for host
– Low [glucose] leads to increase in [cAMP].
– Increased [cAMP] will repress the host gene hflA.
– Less HflA (a protease) leads to less degradation of the
CII protein.
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MOLECULAR GENETIK
ANALYSIS
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Material Genetik
Cetak biru dari semua jenis makhluk hidup sebagian besar berupa DNA
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Untai tunggal DNA adalah rangkaian nukleotida dimana untai baru
selalu dilekatkan pada posisi 3`
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SEKUENSING
Prinsip Sekuensing
Sintesis DNA in vitro dengan DNA Polymerase dan penggunaan 2`, 3`
dideoxynucleotida.
Hilangnya molekul 3`-OH menyebabkan berthentinya reaksi sintesis untai DNA pada
point dimana molekul dideoxynukleotida disintesis, karena tidak ada reaksi yang bisa
dilakukan tanpa adanya molekul tersebut
Sintesis untai DNA baru diawali pada posisi spesifik dengan menggunakan primer
yang menempel pada ujung 3` pada daerah yang akan disekuensing
Reaksi sintesis DNA menggunakan deoxynucleotida dan dideoxynucleotida akan
menghasilkan beberapa fragment DNA yang memiliki residu nukcleotida yang serupa
Perbedaan ukuran fragment DNA dapat dianalisis dengan polyacrilamide gel
elektroforesis
Dua Teknik Sekuensing:
Metode Enzymatic (Metode Sanger)
Metode Degradasi Kimiawi (Metode Maxam - Gilbert)
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DNA Utas tunggal
5`
3`
Primer
3`
5`
4 dNTP
DNA Polymerase
ddTTP
ddCTP
ddGTP
ddATP
DNA baru
dideoxynukleotida
Primer
Sekuensing DNA Hasil PCR
Kloning fragment PCR pada vektor
Sekuensing PCR fragmen dengan Primer Universal yang komplemen
UniversalPri
mer
dengan vektor
Vektor
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C
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Sekuensing Fragmen DNA berukuran panjang
Kloning fragment DNA pada vektor
Sekuensing PCR fragmen dengan Primer Universal yang komplemen dengan vektor
Sekuensing dengan penyusunan primer baru yang kompleme dengan hasil sekuensing dengan
primer universal
Vektor
UniversalPri
mer
Vektor
Primer 1
Primer 2
Vektor
Primer 3
Vektor
Vektor
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Sekuensing Fragmen DNA Secara Random
Purifikasi fragment DNA
Digesti Fragmen DNA dengan sonikasi atau enzim restriksi
Ligasi Fragment DNA pada Vektor
Sekuensing DNA dengan universal primer
Alignment sekuen fragmen DNA dari DNA target
Purifikasi
DNA
Digesti
UniversalPri
mer
Ligasi dan
Sekuensing
Alignment
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