The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes,
critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The
mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety
of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates,
explained in current models by a combination of 3D and 1D diffusion. Here, we describe the combination of optical
tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the
opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in
other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence.
We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular
interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA
interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the
kinetics of both short- and long-lived interactions of LacI along the DNA template (from ∼100 μs to tens of seconds), as
well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions
observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate
DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of
DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time
scales not accessible to other single-molecule methods. This improvement in time resolution provides also important
means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational
changes occurring upon target recognition.
Here we report the effect of DNA tension on lac repressor 1D-diffusion through a combination of single-molecule
localization and optical trapping. The diffusion coefficient shows a parabolic dependence on DNA tension.
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