Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson

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