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.

Lysis Lysogeny

UV Induction

+

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

oR

Pint oL PL PRM PR PRE PR‘tR3

tL1 tR1 tR2 t6S

attint

xisred

gam

cIII N cI cro cII O P Q S R A…J

CIII CII

CI

+

Repressor

PRE = promoter for repressionestablishmentInt

tint

CII

Lysogeny: Repressor turns off transcription

oR

Pint oL PL PRM PR PRE PR‘tR3

tL1 tR1 tR2 t6S

attint

xisred

gam

cIII N cI cro cII O P Q S R A…J

CI

Repressor

PRM = promoter for repressionmaintenance

CI

CIActivated by Repressorbinding to oR1 & oR2

operators overlap promoters

oR1oR2oR :

TTGACT GATAAT-10-35

ATAGAT 5’TTAGAT 5’-10 -35

oR3

cro N

PR

PRM

Repressor structure

operator

N-terminus: DNA binding; Helix-Turn-Helix motif

C-terminal domain: protein-protein interaction; dimerization and cooperativity

Connector

operator operator

oR1oR2

repressor is a dimer; monomer has 236 amino acids.

repressor can bind cooperativelyto operator sub-sites.

-lachybrid genes

lac p, o cI pR , OR lacZ

Place cI gene under lac control. Use lacZ as a reporter.

E. coli with lac repressor, no lacZ.

Control amount of repressor by [IPTG].

See effect of repressor by -galactosidase activity

321

Repressor stimulates transcription from PRM

lac p, o cI pRM , OR lacZ

-galactosidase

[IPTG]

repressor

123

repressor at oR1 and oR2 stimulates transcription from pRM.

Binding of repressor blocks transcription from pR but activates pRM

oR1oR2

-10-35

-10 -35

oR3

cro N

PR

PRM

2 dimers of Repressor, bound cooperatively

RNA Pol

= operator

-10-35 = promoter

Events at initiation of

transcription

Abortive initiation assay

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

R + P RP RPoATP + UTP*

ApUp*U

kf

kr

KB

Abortive transcripts

[ApUp*U]

time

lag

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

Lag time

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

Lag time = KB kf

1

kf

1x +1

[R]

1

[R]Y-intercept = kf

1

Slope = KB kf

1

Effect of wild-type and pc mutant λ repressors on activity of PR & PRM

Effect of Operator Mutations on Transcriptional Control of PR&PRM

OR1-OR2+OR3- OR1+OR2-OR3+

Effect of λ-pc mutations on KB and k2

Architecture of λOR

Mutations in the Activating Region ofλ Repressor

Glu

Glu

Glu

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

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

promoter

A model for interaction of the δ subunit of RNA polymerase with λ repressor

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