Lysogeny maintenance: a matter of looping

Laura Finzi

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

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 repressor auto-regulation (or how to maintain the perfect

concentration)

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

signal

Motion amplitude, (nm)

Time (s)

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)

Labview routine

DIC imagex and y coordinates

Drift

subtraction

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

-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

DNA fragments used in the TPM measurements

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

-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

DNA fragments used in the TPM measurements

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.

Del OL1 Del OL1,2 Del OL1-3

Effect of progressive deletions of the OL operators

control control control20 nM 100 nM 20 nM 100 nM20 nM 100 nM40 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

-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

DNA fragments used in the TPM measurements

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

Loop probability analysis: loop size effect

20 nM 100 nM 100 nMcontrol control

302 bp

control

C

t

100 nM

;

U

U

U

L

L L

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

tttttC )()()(

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( aad

)1( F

dND

2

0

1

0 exp)1(exp1t

at

aF

2

0

1

0

2211

exp)1(exp

exp1

exp

t

at

a

tata

WNpdf

Summary of wild type lifetimesUnloop

CI a u1 u2 N χ2

50 0.35(.02) 12.0(1.0) 92.6(5.7) 330 1.0

100 0.47(.04) 6.0(2.4) 39.0(9.8) 643 1.6

200 0.72(.05) 9.7(0.7) 34.0(5.2) 1016 2.1

400 .73(.8) 8.0(8.0) 20(40) 651 3.5

Loop

CI a L1 L2 N χ2

50 .80(.03) 2.8(.3) 31.8(5.0) 282 1.2

100

200 .64(0) 4.7(.1) 32.9(1.2) 927 1.8

400 .67(0) 3.6(.1) 36.8(1.4) 671 1.4

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.

64 49

1

6

9

18

2

9

1

6

6

6

9

18

6

9

6

1

Possible configurations

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.

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.

Effect of [CI] on the equilibrium

constant of the looping reaction (Kloop)

Kloop = Dloop/Dunloop = total time spent in the looped config./total time spent in the unlooped configuration

)1( F

dND

Dwell times distributions for unlooped and looped states in the presence of 200 nM CI and 10,000X competitor

DNA

Single exponential

Point-mutated operators

1 2 3 3 2 1

O1-

O2-

O3-

L R

x

x

xx

x

x

Probability distribution of <> in mutated DNA fragments

wt-DNA: 6 operators

mutated DNA: 4 operators available

Lifetimes: oL1-oR1-

Lifetimes: oL2-oR2-

Lifetimes: oL3-oR3-

Summary of mutant lifetimesUnloop [CI] = 100 nM

a u1 u2 N χ2

wt 0.47(.04) 6.0(2.4) 39.0(9.8) 643 1.6

oL1-oR1- .56(.05) 6.0(1.7) 72.6(17.8) 197 1.2

oL2-oR2- .57(.03) 17.0(.8) 98.5(10.0) 377 0.7

oL3-oR3- .68(.07) 16.3(1.6) 96(36) 231 0.9

Loop [CI] = 100 nM

a L1 L2 N χ2

wt .66(0) 6.9(.2) 56.7(3.5) 576 2.0

oL1-oR1- .82(.08) 3.6(1.0) 15.5(9.7) 170 1.2

oL2-oR2- .86(.03) 3.3(.6) 18.1(9.8) 302 1.6

oL3-oR3- / 8.1(.4) 166 0.9

Interpretation for O3- single exponential

O3- xx

x

x

O1-xx

xx x

x

xxx

x xxO2-

L 1 2 3 R 3 2 1R

L

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

RT

Gs

RT

Gs

i

unlooped j

looped i

eq

j

j

i

i

Population probability and estimation of ∆Gloop

GL1 = GL1, binding

GL1,2 = GL1, binding + GL2,binding + GL1,2,coop

GLoop1,2 = GL1, binding + GL2,binding + GL1,2,coop +

GR1, binding + GR2,binding + GR1,2,coop + Gloop octamer

∆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-

pi,

loop

/

p i, u

nloo

ped

Dodecamer precursor

L R 1 2 3 3 2 1

Conclusions

1) CI mediates a loop between the L and R region of 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

0s 50s 100s

Time

250n

m+ 0.03+ 0.03

00

- 0.015- 0.015

- 0.03- 0.03

- 0.06- 0.06

Gal repressor requires supercoiling

Lia et al, PNAS 2003

Hat curve for wt DNA fragment

Fragment length: ~ 11,000 bp

Force-jump experiments yield loop lifetime

0.64 microns

Shorter loop shows transitions

Fragment length: ~ 4900 bpLoop size: 393 bp

Force and supercoiling oppose looping

For

ce

Negative supercoiling

Conclusions

1) We observed CI-mediated wild type loop formation and measured loop

2) We can measure the effect of tension and supercoiling on a short CI-mediated loop

3) Level of supercoiling is critical for the stability of the loop

4) More experiments are needed!!!!

• 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