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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


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.


UV Induction

Lysogeny

Lysis


+

+

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


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 promoter (open)

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 =


Effect of wild type and pc mutant repressors on activity of p r p rm
Effect of wild-type and pc mutant promoter (open)λ repressors on activity of PR & PRM


Effect of operator mutations on transcriptional control of p r p rm
Effect of Operator Mutations on Transcriptional Control of P promoter (open)R&PRM

OR1+OR2-OR3+

OR1-OR2+OR3-


Effect of pc mutations on k b and k 2
Effect of promoter (open)λ-pc mutations on KB and k2


Architecture of o r
Architecture of promoter (open)λOR


Mutations in the activating region of repressor
Mutations in the Activating Region of promoter (open)λ Repressor

Glu

Glu

Glu


Mutations in the promoter (open)δ subunit of RNA polymerase that interfere w/λ repressor-mediated activation of PRM transcription


Effect of mutations in the promoter (open)δ subunit of RNA polymerase on activator-dependent and independent transcription of the lac promoter


A model for interaction of the subunit of rna polymerase with repressor
A model for interaction of the promoter (open)δ subunit of RNA polymerase with λ repressor



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