Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Abstract

The paper describes 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. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. Video LinkThe video component of this article can be found at

Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching

“…In a fully extended DNA structure, the distance between the two O1 operators corresponds to ∼103 nm (304 bp × 0.34 nm/bp) and the distance between O3 and the two O1 operators to ∼72 and ∼31 nm. A custom-built multichannel flow system is used to sequentially trap two beads in the bead channel, attach a single DNA molecule between the two beads in the DNA channel, and move the dumbbell in the buffer channel for measurement (Figure 1B and ‘Materials and Methods’ section) ( 17 ). After testing the presence of a single DNA molecule by force-extension analysis, DNA is slightly stretched (F ∼3 pN) and positioned in the proximity of a third bead stuck on the coverslip, containing a single LacI protein (Figure 1A ).…”

“…A laminar flow system was used to incubate biological samples, perform buffer exchanges and to efficiently anchor DNA molecules between the two optically trapped beads (1.07 μm streptavidin-coated polystyrene beads, Spherotech). These protocols are described with great detail elsewhere ( 17 ). The multichannel flow chamber (capacity of ∼50 μl) was assembled on top of a coverslip previously coated with silica beads (1.21 μm Bangslabs) and nitrocellulose as described previously ( 21 , 22 ).…”

“…Briefly, the experimental setup comprises an inverted optical microscope combined with double optical tweezers and fluorescence microscopy with single-molecule sensitivity. The apparatus is stabilized to less than 1 nm with both passive and active stabilization ( 17 , 25 ). The active nm-stabilization system uses the bead with the Lacl molecule as a landmark to determine the sample coordinates and correct for thermal drifts and low-frequency noise by moving piezo translators.…”

Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI

“…Variants of LacI bearing Cys-to-Ala substitutions at all three native cysteine positions have previously been used in studies of the LacI-induced DNA looping [ 70 ] and LacI diffusion on flow-stretched DNA [ 71 ], indicating that such native cysteine-free variants are functional. As opposed to the LacI variants in these studies, our “LacI Cys-Lite ” lacks the C-terminal tetramerization helix.…”

Data on the target search by a single protein on DNA measured with ultrafast force-clamp spectroscopy

“…The data presented here is acquired using ultrafast force-clamp spectroscopy [3] and shows a much higher spatial and temporal resolution and much longer observation time compared to analogous single molecule studies using fluorescence microscopy [2] , [4] , [5] , [6] , [7] . Therefore, the data might contain valuable information on rapid (ms) conformational changes of the lactose repressor protein (LacI) during its interaction with cognate and non-cognate DNA sequences, as well as conformational changes occurring on a time scale of several minutes [8] .…”