Lysogeny maintenance: a matter of looping

Laura Finzi

λ is a temperate phageTwo possible modes LYSOGENIC MODE (passive replication)

LYTIC MODE (active replication)

EFFICIENT REGULATION OF GENIC EXPRESSION

Lysogenic mode

Lytic mode

replication

0 minutes

45 minutes

30 minutes

Lytic cycle

Ptashne M., 1992, “A genetic switch”, Cambridge, MA: Blackwell Scientific Pubblications and Cell Press

λ CI protein acts both as a transcriptional activator and as a repressor in the maintenance of the lysogenic cycle

λ repressor (CI) is responsible for maintenance of lysogeny

The occupancy of OR3 by CI and, consequently, the mechanism of negative autoregulation, depend on the interaction among CI molecules bound to the OL and OR regions, about 2.4 kbp apart

Revet B. et al. Current Biology 9:151-154, 1999. / Dodd I.B. et al. Genes and development 15:3013-3022, 2001. /

Dodd I.B. et al. Genes and development 18:344-354, 2004.

Loop-based model of the λ repressor auto-regulation (or how to maintain the perfect concentration)

TPM-Protein-induced dynamic DNA looping produces a telegraphic-like signal

ρ⊥

Mot

ion

ampl

itude

, ρ ⊥

(nm

)ρ⊥

ρ⊥

Time (s)ρ⊥

D. Schafer et al. Nature 352:444, 1991 / L. Finzi & J. Gelles Science 267:378, 1995

P. Nelson, et al., “Tethered Particle Motion as a Diagnostic of DNA Tether Length”, The Journal of Physical Chemistry B, 110, 17260, 2006

Data aquisition and analysis (1)

Labviewroutine

DIC imagex and y coordinates

Driftsubtraction

x’ and y’ coordinates of the anchor point

ρ┴(t) = [(x(t)-x’)2+(y(t)-y’)2]1/2

Plot of ρ┴ over time

P. Nelson, et al., “Tethered Particle Motion as a Diagnostic of DNA Tether Length”, The Journal of Physical Chemistry B, 110, 17260, 2006

OL1 OL2 OL3 OR3 OR2 OR1

302 – 306 – 1051- 2317 bp

OL2 OL3 OR3 OR2 OR1

OL3 OR3 OR2 OR1

DelOL1

DelOL1,2

DelOL1-3

DigBio

OR3 OR2 OR1

Bio

Bio

Bio

Dig

Dig

Dig

DNA fragments used in the TPM measurements

-Verified loop formation in the DNA mediated by CI bound to the OLand OR regions

-Determined the relative importance of the three OL operators in loop formation

-Determined the effect of the distance between the OL and OR regions

C. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

Wt loop formation and breakdown

OR3 OR2 OR1

OL1 OL2 OL3 OR3 OR2 OR1

302 – 306 – 1051- 2317 bp

OL2 OL3 OR3 OR2 OR1

OL3 OR3 OR2 OR1

DelOL1

DelOL1,2

DelOL1-3

DigBio

Bio

Bio

Bio

Dig

Dig

Dig

DNA fragments used in the TPM measurements

-Verified loop formation in the DNA mediated by CI bound to the OL and OR regions

-Determined the relative importance of the three OL operators in loop formation

-Determined the effect of the distance between the OL and OR regionsC. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

Del OL1 Del OL1,2 Del OL1-3

Effect of progressive deletions of the OL operators

control 20 nM 100 nM40 nM control 20 nM 100 nM control 20 nM 100 nM

C. Zurla, et al., J.P.C.M. 18, S225-S234, 2006.

OL1 OL2 OL3 OR3 OR2 OR1

302 – 306 – 1051- 2317 bp

OL2 OL3 OR3 OR2 OR1

OL3 OR3 OR2 OR1

DelOL1

DelOL1,2

DelOL1-3

DigBio

OR3 OR2 OR1

Bio

Bio

Bio

Dig

Dig

Dig

DNA fragments used in the TPM measurements

-Verified loop formation in the DNA mediated by CI bound to the OL and OR regions

-Determined the relative importance of the three OL operators in loop formation

-Determined the effect of the distance between the OL and OR regions

C. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

1051 bp

2317 bp

100 nM 100 nMcontrol control

Loop probability analysis: loop size effect

20 nM

302 bp

control

C

Δt100 nM

;

U

U

U

L

C. Zurla, et al., J.P.C.M. 18, S225-S234, 2006.

L

L

tttttC )()()( Δ+∗=Δ ρρ

PRM expression titration

Looping Titration (CI nM)

20

50 400

200

1000

Dwell times distributions for unlooped and looped states at [CI] = 50 and 200 nM

21 )1( ττ aad −+=

)1( FdND−

=

⎟⎟⎠

⎞⎜⎜⎝

⎛−−−⎟⎟

⎠

⎞⎜⎜⎝

⎛−−=

2

0

1

0 exp)1(exp1ττtataF

⎟⎟⎠

⎞⎜⎜⎝

⎛−−+⎟⎟

⎠

⎞⎜⎜⎝

⎛−

⎟⎟⎠

⎞⎜⎜⎝

⎛−

−+⎟⎟

⎠

⎞⎜⎜⎝

⎛−

=

2

0

1

0

2211

exp)1(exp

exp1exp

ττ

ττττtata

tata

WNpdf

Summary

Loop formation probability increases with CI protein concentration.There are multiple unlooped and looped species.

Hypothesis:1) Closure of the loop can be mediated by DNA/CI complexes containing

different numbers of CI dimers (different occupation/loading levels) each with different stability.

SummaryLoop formation probability increases with CI protein concentration.There are multiple unlooped and looped species.

Hypothesis:1) Closure of the loop can be mediated by DNA/CI complexes containing different

numbers of CI dimers (different occupation/loading levels) each with different stability.

OL3 OL2 OL1

PRM OR3 OR2 OR1

PR

PL

OL3 OL2 OL1

PRM OR3 OR2 OR1

PR

PL

8mer – pRM is transcribed 12mer – all the promoters are repressed

• Competition experiments,• Measurements with point-mutated operators.• DHMM analysis (J.F. Beausang et al., BJ-BLetters, DNA looping kinetics analyzed using DHMM, 2007) may help characterize hidden intermediates.

Dwell times distributions for unlooped and looped states in the presence of 200 nM CI

and 10,000X competitor DNA

Single exponential

Point-mutated operators1 2 3 3 2 1

O1-

O2-

O3-

L R

xx

xx

xx

Probability distribution of <ρ> in mutated DNA fragments

wt-DNA: 6 operators

mutated DNA: 4 operators available

Lifetimes: oL1-oR1-

Lifetimes: oL2-oR2-

Lifetimes: oL3-oR3-

Estimation of ∆Gloop

For each measured looping equilibrium an expression can be developed in terms of [CI] and free energy, leaving looping free energies as fit parameters.

[ ]

[ ]

looping i,itycooperativ i,binding i,

2

2 ionconfigurat ofy probabilit

GGGG

eCI

eCIp

p

pK

i

RTG

s

RTG

s

i

unlooped j

looped ieq

j

j

ii

Δ+Δ+Δ=Δ

==

=

∑

∑∑

Δ−

Δ−

∆Gloop

∆GLOOP tetramer: 1 kcal/mol∆GLOOP octamer: 0.8 kcal/mol∆GLOOP dodecamer: -0.5 kcal/mol

Black: wt DNA,Blue: O1

-,Red: O2

-

Green: O3-

Σpi,

loop

/ Σp i

, unl

oope

d

Conclusions1) CI mediates a loop between the L and R region of λ DNA

2) OL1 and OL2 are critical for loop formation

3) Wild type loop is quite dynamic

4) Probability of loop formation increases with CI concentration

5) There are multiple possible looped species

6) Competition and point mutated operators (O3-) allow detection of octamer-mediated loop

7) Population probability analysis indicates that octamer-mediated loop is quite unstable and suggests a decamer might be the precursor

• Emory University• Chiara Zurla• Carlo Manzo• Laura Finzi• David Dunlap

Contributors

NCI, NIH•Dale Lewis•Sankar Adhya

University of PennsylvanyaJohn BeausangPhil Nelson

Support: HFSP, Emory University Research Council