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Bacteriophage lambda (l). Transcriptional switches can regulate cellular decisions. Lysis or Lysogeny. Lysis : Infection by phage produces many progeny and breaks open (lyses) the host bacterium

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bacteriophage lambda l

Bacteriophage lambda (l)

Transcriptional switches can regulate cellular decisions

slide2

Lysis or Lysogeny

  • Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium
  • Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively
    • No progeny
    • No lysis of the host
    • Can subsequently lyse (lysogeny)
  • Bacteriophage lambda can do either.
slide3

UV Induction

Lysogeny

Lysis

slide4

+

+

CIII

CII

CII

gam

int

red

N

cI

cro

cII

O

P

Q

S

R

A…J

att

xis

cIII

Pint

tL1

PL

oL

PRM

PR

tR1

PRE

tR2

PR‘

t6S

tR3

oR

CI

Int

Lysogeny: CII and CIII stimulate expression of cI to make repressor

tint

PRE = promoter for

repression

establishment

Repressor

slide5

gam

int

red

N

cI

cro

cII

O

P

Q

S

R

A…J

att

xis

cIII

Pint

tL1

PL

oL

PRM

PR

tR1

PRE

tR2

PR‘

t6S

tR3

oR

CI

CI

CI

Lysogeny: Repressor turns off transcription

PRM = promoter for

repression

maintenance

Repressor

Activated by Repressor

binding to oR1 & oR2

l operators overlap promoters
l operators overlap promoters

oR :

oR3

oR2

oR1

PR

-35

-10

TTGACT

GATAAT

cro

N

TTAGAT 5’

ATAGAT 5’

-10

-35

PRM

repressor structure
Repressor structure

l repressor is a dimer; monomer has 236 amino acids.

l repressor can bind cooperatively

to operator sub-sites.

l lac hybrid genes
l-lachybrid genes

PlacelcI gene under lac control.

Use lacZ as a reporter.

lac p, o

l cI

l pR , OR

lacZ

321

Control amount of l repressor by [IPTG].

E. coli with lac repressor,

no lacZ.

See effect of l repressor

by b-galactosidase activity

repressor stimulates transcription from p rm
Repressor stimulates transcription from PRM

lac p, o

l cI

l pRM , OR

lacZ

123

b-galactosidase

l repressor

[IPTG]

l repressor at oR1 and oR2 stimulates transcription from pRM.

binding of repressor blocks transcription from p r but activates p rm
Binding of repressor blocks transcription from pR but activates pRM

PR

-35

-10

2 dimers of

Repressor,

bound

cooperatively

oR3

RNA Pol

cro

N

oR2

oR1

-10

-35

= operator

PRM

= promoter

-35

-10

abortive initiation assay

Let R = RNA polymerase, P = promoter (closed), and Po= promoter (open)

ATP + UTP*

kf

KB

RP

RPo

R + P

kr

Abortive transcripts

ApUp*U

[ApUp*U]

lag

time

Abortive initiation assay
measure k f and k b from lag time vs 1 r

1

1

1

1

1

1

KB kf

KB kf

[R]

[R]

kf

kf

Measure kf and KB from lag time vs. 1/[R]

Lag time in abortive initiation assay is inversely proportional to [R].

Lag time =

x

+

Lag time

Slope =

Y-intercept =

slide19
Mutations in the δ subunit of RNA polymerase that interfere w/λ repressor-mediated activation of PRM transcription
slide20
Effect of mutations in the δ subunit of RNA polymerase on activator-dependent and independent transcription of the lac promoter
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