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  • Advanced Microscopy Techniques III
  • SPIE Proceedings (Optica Publishing Group, 2013),
  • paper 87970F

Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

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Abstract

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.

© 2013 SPIE

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