Lac Repressor-DNA Interactions assessed by Ultrafast Force-Clamp Spectroscopy

Abstract

We recently developed an ultrafast force-clamp laser trap technique [Capitanio et al., Nature Methods 9,1013-1019(2012)] that allows probing, under controlled force, both long- and short-lived biomolecular interactions (100μs to tens/hundreds of seconds), as well as sub-nanometer conformational changes occurring upon bond formation. Here, we show the application of our method to the study of lactose repressor (LacI). Our results show two kinetically well-distinct populations of interactions, which clearly represent strong interactions (targeting the two operators located 100nm apart from each other: long events in the figure) and fast scanning of LacI along non-cognate DNA (during target-search: short events in the figure). Our results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA molecule and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other 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.This research is funded by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX.View Large Image | View Hi-Res Image | Download PowerPoint Slide