Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

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.

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