Bacteriophage Lambda

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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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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



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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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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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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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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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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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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


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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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


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


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



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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



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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



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Dimerizationis lost

Repressor drops offoperators

Transcription initiatedfrom PL and PR

Lytic cyle



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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



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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)



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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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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



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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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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


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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Bacteriophage



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p g y y g p


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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)


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


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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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


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`


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



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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 .



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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

Bacteriophage Lambda

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