Mapping Lac Repressor Interactions along DNA with Ultra-Fast Optical Tweezers

Transcription factors and DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA [1]. One important issue in the field of protein-DNA interaction is the understanding of how proteins interact with non-cognate DNA sequences and how they find the sequence of interest along the DNA. We developed a system that permits to detect protein-DNA interaction with sub-ms temporal resolution and nanometer spatial precision [2]. Such precision allows us to discriminate between short events (milliseconds), which are non-specific, and long events (tens of seconds) considered to be highly specific, and to determine the precise location along the DNA where they occur. We chose a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We used a DNA molecule containing two copies of the O1 and one of the O3 operator placed at known distances and we obtained a map of the long interactions along the DNA molecule, corresponding to the position of the two O1 operators. Short interactions were instead spread along the whole DNA molecule but occurred with higher probability in correspondence of the operators and in their proximity. Dissociation of both classes of interactions was highly accelerated by an external load. Measurements performed in the presence of IPTG, a mimic of the inducer allactose, resulted in increased dissociation from the lac operators. [1] Monico, C. et al., Int. J. Mol. Sci. 14, 3961-3992 (2013). [2] Capitanio, M. et al., Nature Methods, 9, 1013-1019 (2012)