Protein DNA Interaction Measured with Ultra Fast Force Clamp Optical Tweezers

The lac operon is a well known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). LacI and other 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. We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with two different DNA constructs: a construct with two copies of the O1 operator separated by 300 bp and a construct containing the native E.coli operator sequences. Our measurements (see Figure) show at least two classes of LacI-DNA interactions: long (tens of s) and short (tens of ms). Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operators (long events) and transient interactions with nonspecific sequences (short events). Moreover, we observe continuous sliding of the protein along DNA, passively driven by force applied with the optical tweezers.View Large Image | View Hi-Res Image | Download PowerPoint Slide