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To Loop, or Not to Loop, That is the Question. Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson. Why should you care about Looping?. Transcriptional Regulation - Difference Between Life and Death VDJ Recombination - Difference Between Life and Death - PowerPoint PPT Presentation
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Geoffrey Lovely, Sungwook Woo, and David Schwab,
with help of Stephanie Johnson
To Loop, or Not to Loop,That is the Question.
Why should you care about Looping?
• Transcriptional Regulation- Difference Between Life and Death
• VDJ Recombination- Difference Between Life and Death
• Evolution- Difference Between Life and Death
Lac I ( Repressor) Operators (O1> O3> O2)
Lac Z(ß-Galactosidase) Lac Y(Permease)
Lac A (Transacetylase) Promoter (PI and Plac)
Lac Z
Lac Y
Lac A
O2 O1
O3
PLac
1080
LAC OPERON
LacI
92 401 2671 1251 609 bps
Oehler et al. 1994
Lac Repressor
Tetramer bound to two operators
To Simplify
=
Swigon et al. 2006
O2 O1 O3
Lac Operon in the Presence of Glucose
LacI
• 1) In the Presence of Glucose Lac Repressor is produced
• 2) Lac Repressor binds to operator halts PLac expression
Note: In solution the Repressor forms tetramers
mRNA
Protein Monomer of Repressor
Tetramer of Repressor
Glucose
(Ruben et al. 1997)
All Possible Binding States
O1 O3 O2
O1 O3 O2
O1 O3 O2
O1 O3 O2 O1 O3 O2
O1 O3 O2 O1 O3 O2
One Tetramer
Two Tetramers
Three Tetramers
All Possible Looped States of Lac Repressor
One Tetramer
Two Tetramers
We use beads (~0.5 m) to observe single DNA molecules.
Experimental Scheme
Lin Han’s thesis
Lac
The effective length of the DNA strand will get shorter when looped.
Hence, the lateral scope of the movement of the bead will get smaller.
The movement of each bead indicates the state of each single DNA molecule!
Experimental Scheme
DoubleSided Tape
Glass Slide
Cover Slip
Input Output
Experimental Scheme
Experimental Scheme
Results & Discussion
Raw data RMS
Raw data RMS
Results & Discussion
Concentration (M)
Pro
babi
lity
1X10-12 1X10-8 1X10-4
1
0
Concentration Dependence
Histograms of RMS Data
No Lac 100 fM
100 pM 100 nM
O1-O3
No Loop
O1-O2
Conclusion
• Data analysis: Gaussian filter, Hidden Markov
• Move/delete operators, relative phasing
• Measure looping free energies
• More detailed concentration dependence
• Kinetics of transitions
Future work
Results & Discussion
