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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
129
62-POS
Board 31
Measuring the Orientation of Proteins Sliding on DNA
Emil Marklund
, Elias Amselem, Kalle Kipper, Magnus Johansson, Sebastian Deindl, Johan Elf.
Uppsala University, Uppsala, Sweden.
Many transcription factors and restriction enzymes slide on DNA when searching for their
specific target sites. Indirect evidence suggests that some of them also rotate around the DNA in
a helical path while sliding. Little is however known about the orientation of the proteins while
sliding and how it changes in time. To bridge this gap, we are measuring the orientation of
the
lac
repressor labeled with a fluorophore that is rigidly attached to the DNA binding domain .
In our experiments we measure polarization of the light emitted from single transcription factor
molecules when they are bound specifically or sliding on flow stretched DNA. The polarization
data acquired is shifted towards the direction of DNA with relatively small changes in the signal
over several seconds when using camera tracking with an integration time of ~100 ms. This
shows that the transcription factor has a preferred and maintained orientation on this timescale.
For transcription factors sliding on DNA, very little polarization preference is observed in the
direction that would be averaged out by rotation of the fluorophore around the DNA. We have
also studied the sliding process at the faster timescale expected for one revolution of the protein
around the DNA. For this we have developed a tracking confocal microscope with real time
position feedback and a photon time tagging resolution of 5 ns. By doing fluorescence
correlation spectroscopy we get a decay time in the range expected for transcription factor
molecules rotating around the DNA while sliding. Taken together these observations strongly
support the model of rotational sliding for the
lac
repressor.