Understanding Lytic Infection and Lysogeny

Divergent transcription.

Divergent transcription.

• Gene A Gene B

strong weak

Concentration Dependent occupancy

• 1950’s Andre Lwoff, Francis Jacob, Jacques Monod at Institute Pasteur.

• E. coli irradiated… 90 minutes later cells lyse (burst)

• Bacteriophage Lambda.

• Switching between two states of the virus:

• Latent (lysogeny) vs. lytic

• Most phage interact with cells in one way - they take over the

machinery of macromolecular synthesis to produce a large number of viral particles

•

• The end result is lysis

•

• There is a highly ordered sequence of events leading to lysis

•

• This entire process is called a lytic infection

• In some infections, the phage can co-exist with the cell

• The phage can go lytic under certain conditions

• This is called LYSOGENY - which means the potential for lysis

Lytic Lysogeny

• Lambda is an Obligate parasite (must inject DNA into bacterium to multiply).

• 45 minutes after infection bacterium lyses and releases 100 progeny phage.

• UV light induces previously inert phage

genes and lytic growth.

• In a lysogen there is only one gene is on that encodes the Lamda repressor.

• Both a positive and a negative regulator of

gene expression.

Components of the Switch

17bp

O_R1 O_R2

O_R3 cI

cro P_RM

P_R

Components of the Switch

17bp

O_R1 O_R2

O_R3 cI

cro P_RM

P_R

cI: there are other genes cII cIII

Cro: control of repressor and other genes PRM: promoter of repressor maintenance

• Polymerase from bacterial host

• The two promoters are never occupied simultaneously

• Repressor 236 aa protein

1-92 40 132-236

N C

Repressor

N C N C N C N C

17 bp N C

Cro

All cro in cells exist as dimers Because of very high affinity 66 aa

Hypothetical examples

O_R1 O_R2

O_R3

OFF cro P_RM

P_R

NC NC

ON

Normally Rep can never bind Or2 without Or1

O_R1 O_R2

O_R3

OFF cro P_RM

NC NC

OFF

So Repressor has both positive and negative role

O_R1 O_R2

O_R3

ON cro P_RM

P_R

NC NC

OFF

In the cell

O_R1 O_R2

O_R3

O_R1 O_R2

O_R3

R R

R

R R

>90%

<10%

Cooperative binding

O_R1 O_R2

O_R3

NC NC

N C

N C N

C N

C

N C

N C N

C N

C N

C

N C Increasing

Rep

Concentration

strong weak

Concentration Dependent occupancy

Cooperative binding

O_R1 O_R2

O_R3

NC NC

N C

N C N

C N

C

Linker is flexibleso Repressor at

Or2 can contact Rep at Or1

X

N C

N C N

C N

C

Affinity of Rep to OR2 and OR3 increases 5 fold above their intrinsic affinity

Flipping the switch

N C

N C N

C N

C

OFF ON

In a lysogen Rep at OR1 and OR2 stimulates Prm and turns off Pr.

Rep is constantly being synthesized as cells grow and divide, and the Cro gene remains silent.

Flipping the switch

N C

N C N

C N

C

OFF ON

In a lysogen if cell division stops Rep concentraion increases, binds OR3, turns off its own transcription.

N C

N C N

C N

C

OFF OFF

N C

N C

Flipping the switch

In a lysogen if cell division starts Rep concentration decreases, binds OR3, turns on its own transcription.

N C

N C N

C N

C

OFF OFF

N C

N C

N C

N C N

C N

C

OFF ON

Octameric CI:OR-OL complex increases the affinity of CI for OR3 by allowing a CI tetramer to link OR3 and the third operator at OL, OL3

Flipping the switch

RecA, a bacterial UV response protein cleaves Rep in the Linker between Ala and Gly.

Rep falls off of the operator without C terminal dimerization domain.

N C N C

RecA UV

Active RecA

Flipping the switch

N C N C

RecA

DNA

Active RecA

SOS response (cleaves lexA protein) Also cleaves lambda repressor

RecA bound to ssDNA stimulates the cleavage of CI monomers removing CI's ability to bind DNA cooperatively and reducing the activity of CI to a point where the lytic promoters become derepressed

Repressor

N C N C N C N C

17 bp N C

When Rep concentration decreases

O_R1 O_R2

O_R3

ON

P_RM P_R cro

OFF cI

When Rep concentration decreases

O_R1 O_R2

O_R3

OFF cro

OFF cI

Cro binds non-cooperatively to Pr and Prm.

Cro first turns off cI (Prm) then turns off its own transcription (Pr)

When Rep concentration decreases

ON OFF

O_R1 O_R2

O_R3

ON cro

OFF

cI

OFF OFF

OFF 3 OFF

1

2

Increasing Cro Concentration

Rep conc Repression

99.7% repression

lysogen

10 100 1000

Cooperativity between repressors causes rapid activation Dimerization and DNA binding

Cooperative binding

O_R1 O_R2

O_R3

NC NC

N C

N C N

C N

C

Linker is flexible so Repressor at

Or2 can contact Rep at Or1

Rep conc Repression

99.7% repression

lysogen

10 100 1000

Pr ON

Pr OFF

2x 5x

Switch Stability

Rep conc Repression

99.7% repression

lysogen

10 100 1000

Pr ON

Pr OFF

2x 5x Single repressor

operator system

• Autoregulation Or3 causes turn off of Prm.

So that the cells don’t get too high a

concentration of Rep, so that they cannot be activated in response to an inducer

signal like UV.

• If concentration of Rep, is so high that

even cleaved Rep N terminal domain can bind to OR1-2-3, then you can never get activation.

• This (squelching) is prevented because

the affinity of cleaved repressor is low

towards the operator sites.

Helix 2+3 Helix turn helix motif

When rep docks each recognition Helix docks into the major groove of DNA.

Helix 2 helps position helix 3 into groove.

Val from Helix 3 makes contact with ala from helix 2 Helix 2 makes contacts with Pol. (Ile-Ala for Cro)

Gln in helix 3 makes contact with A in operator.

Val from Helix 3 makes contact with ala from helix 2 Helix 2 makes contacts with Pol. (Ile-Ala for Cro)

How does Lamda decide between

lysogeny and lytic cycles?

Cos Reco

Att

cIII N

cI

cro cII

Replication Q

Lysis tail

head

CG

GGGCGGCGACCTCG GCCCCGCGCTGGA

GC

Cohesion happens immediately after injection by ligase

AttP X

AttB

Phage Attachment site

Bacterial Attachment site

Phage

repression of PRM at lysogenic CI concentrations is absolutely dependent on OL. A mutant defective in this CI negative autoregulation forms a lysogen with elevated CI levels that cannot efficiently switch from lysogeny to lytic development

Model for the intact repressor octamer bound to four operator sites

OR1 OR2

The four operator sites represent O_R1 and O_R2 (on the right) and O_L1 and O_L2 (on the left). The structure of the octameric CTD determined in this study is shown in the central portion of the figure, w

The repressor model for gene regulation of the lac system was that the lacI gene makes a product that acts at the operator to turn off lacZ, the gene that encodes the enzyme β- galactosidase. The figure implies that the operator is DNA, a fact established only after the isolation of repressors. Although the lacZ gene is shown expressing its product in the absence of repressor, high levels of expression of lacZ require the activating effect of CAP, a fact only recognized well after the repressor model was proposed (see text).