lysogeny maintenance: a matter of looping laura finzi
DESCRIPTION
Lysogeny maintenance: a matter of looping Laura Finzi. Two possible modes. LYSOGENIC MODE (passive replication). LYTIC MODE (active replication). Lytic mode. Lysogenic mode. replication. l is a temperate phage. EFFICIENT REGULATION OF GENIC EXPRESSION. 0 minutes. 30 minutes. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/1.jpg)
Lysogeny maintenance: a matter of looping
Laura Finzi
![Page 2: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/2.jpg)
is a temperate phageTwo possible modes LYSOGENIC MODE (passive replication)
LYTIC MODE (active replication)
EFFICIENT REGULATION OF GENIC EXPRESSION
Lysogenic mode
Lytic mode
replication
![Page 3: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/3.jpg)
0 minutes
45 minutes
30 minutes
Lytic cycle
![Page 4: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/4.jpg)
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
![Page 5: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/5.jpg)
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)
![Page 6: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/6.jpg)
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
![Page 7: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/7.jpg)
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
![Page 8: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/8.jpg)
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.
![Page 9: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/9.jpg)
Wt loop formation and breakdown
![Page 10: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/10.jpg)
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.
![Page 11: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/11.jpg)
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.
![Page 12: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/12.jpg)
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.
![Page 13: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/13.jpg)
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 )()()(
![Page 14: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/14.jpg)
PRM expression titration
![Page 15: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/15.jpg)
Looping Titration (CI nM)
20
50 400
200
1000
![Page 16: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/16.jpg)
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
![Page 17: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/17.jpg)
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
![Page 18: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/18.jpg)
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.
![Page 19: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/19.jpg)
64 49
1
6
9
18
2
9
1
6
6
6
9
18
6
9
6
1
Possible configurations
![Page 20: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/20.jpg)
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.
![Page 21: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/21.jpg)
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
![Page 22: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/22.jpg)
Dwell times distributions for unlooped and looped states in the presence of 200 nM CI and 10,000X competitor
DNA
Single exponential
![Page 23: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/23.jpg)
Point-mutated operators
1 2 3 3 2 1
O1-
O2-
O3-
L R
x
x
xx
x
x
![Page 24: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/24.jpg)
Probability distribution of <> in mutated DNA fragments
wt-DNA: 6 operators
mutated DNA: 4 operators available
![Page 25: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/25.jpg)
Lifetimes: oL1-oR1-
![Page 26: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/26.jpg)
Lifetimes: oL2-oR2-
![Page 27: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/27.jpg)
Lifetimes: oL3-oR3-
![Page 28: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/28.jpg)
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
![Page 29: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/29.jpg)
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
![Page 30: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/30.jpg)
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
![Page 31: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/31.jpg)
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
![Page 32: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/32.jpg)
∆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
![Page 33: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/33.jpg)
Dodecamer precursor
L R 1 2 3 3 2 1
![Page 34: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/34.jpg)
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
![Page 35: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/35.jpg)
![Page 36: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/36.jpg)
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
![Page 37: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/37.jpg)
Hat curve for wt DNA fragment
Fragment length: ~ 11,000 bp
![Page 38: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/38.jpg)
Force-jump experiments yield loop lifetime
0.64 microns
![Page 39: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/39.jpg)
Shorter loop shows transitions
Fragment length: ~ 4900 bpLoop size: 393 bp
![Page 40: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/40.jpg)
Force and supercoiling oppose looping
For
ce
Negative supercoiling
![Page 41: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/41.jpg)
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!!!!
![Page 42: Lysogeny maintenance: a matter of looping Laura Finzi](https://reader030.vdocuments.pub/reader030/viewer/2022032313/56812bfc550346895d906cf7/html5/thumbnails/42.jpg)
• 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