Magnetic Tweezers Instrumentation: We have used magnetic tweezers to study chromatin assembly and disassembly and RNA transcription.

Magnetic tweezers

surface

magnetic bead

F

DNA

external magnets

F =kBT l/<δx2>

l

θ

F

δx

surface

Instrumental set-up

video camerabeam condenser

hollow bearing with magnet

90x oil objective

square glass cuvette

white light source

computer

external magnet

flow

magnetic bead on a single DNA

objective

x3,y3

distance traveled across screen

x1,y1

x2,y2

projection of bead on screen

video camerabeam condenser

hollow bearing with magnet

90x oil objective

square glass cuvette

white light source

computer

external magnet

flow

magnetic bead on a single DNA

objective

x3,y3

distance traveled across screen

x1,y1

x2,y2

projection of bead on screen

In collaboration with Profs. J. Zlatanova and W. McAllister, we have been able to visualize transcription from a single T7 RNA polymerase.

frames of video image

nucleosomal arrayassembly under constant force

histones and assembly factors flowed in

starting position

histoneoctamers

flow flow stopped

assembly

magnetic bead

DNA

attachment toinner surface

of cuvette

frames of video image

nucleosomal arrayassembly under constant force

histones and assembly factors flowed in

starting position

histoneoctamers

flow

histoneoctamers

flow flow stoppedflow stopped

assemblyassembly

magnetic bead

DNA

attachment toinner surface

of cuvette

flow turned off

Trav

el o

f be

ad a

cros

s sc

reen

(µm

)

Time (s)600

4

8

12

16

200 400 600

4

8

12

16

200 400

Assembly curves in stopped-flow experiments

4 mm (3.3 pN)

Trav

el o

f be

ad a

cros

s sc

reen

(µm

)

Distance between external magnet and cuvette (mm) and corresponding force

(pN)

Time (s)

2 mm (10 pN)2.5 mm (8.8 pN)

3 mm (4.5 pN)

0

2

4

6

500 1000 1500

Step-wise adjustment of force in a single assembly experiment

We are continuing these experiments to study chromatin dynamics, transcription and the mechanism of action of helicases. References: Leuba, S. H., Karymov, M. A., Tomschik, M., Ramjit, R., Smith, P., and Zlatanova, J. Assembly of single chromatin fibers depends on the tension in the DNA molecule: magnetic tweezers study. Proc. Natl. Acad. Sci. USA 100, 495-500, 2003. Zlatanova, J. and Leuba, S. H. Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level. Biochem. Cell Biol. 81, 151-159, 2003. Leuba, S. H., Bennink, M. L., and Zlatanova, J. Single molecule analysis of chromatin. In Chromatin and Chromatin Remodeling Enzymes, Parts A and B. Methods Enzymol. (C. Wu and C. D. Allis, Eds.) 376, 73-105, 2004. Bennink, M. L., Leuba, S. H. and Zlatanova, J. Analysis of protein/DNA interactions by optical tweezers: application to chromatin fibers. In Protein-protein interactions, a

N S

DNA template(topologically-constrained)

RNAimmobilized

elongating RNApolymerase

objective

marker bead

2800 nm magnetic

bead

external magnets

Single-molecule transcription viewed by magnetic tweezers: experiment

marker bead

One full rotation of the bead as the RNA polymerasetranscribes one helical turn of the DNA

molecular cloning manual, 2nd ed. (E. Golemis and P. Adams, Eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor (by invitation) Ch. 19, pp.1-14, 2005. Pomerantz, R. T., Ramjit, R., Gueroui, Z., Place, C., Anikin, M., Leuba, S., Zlatanova, J., and McAllister, W. T. A tightly regulated molecular motor based upon T7 RNA polymerase. Nano Lett. 5, 1698-1703, 2005.