Abstract
Lac Repressor (LacI) is a DNA-binding protein that regulates genes expression by specific binding to its target sequence on DNA. This sequence is found rapidly among millions of base pairs in the genome, presumably via a combination of 3D and 1D-diffusion (following the interaction with non-specific DNA). Here we report diffusion analysis through the combination of single molecule localization and manipulation. For single molecule localization, Atto532 dye has been covalently linked to a single-cysteine mutant of LacI, LacIQ231C. A double optical tweezers system is used for trapping and stretching a single DNA molecule. All components of the experiment (protein, DNA, beads and buffer) are assembled (and/or rapidly exchanged) with a flow-system. This integrated configuration has several advantages: biomolecules are not in the proximity of the glass surface thus preventing possible electrostatic effects; controlled forces can be measured or applied. We characterized LacI 1D-diffusion under different forces applied to the DNA. The figure shows an example of the data obtained in the form of a kymogram.Acknowledgments:The research leading to the results has received funding from Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAXView Large Image | View Hi-Res Image | Download PowerPoint Slide
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