Abstract
DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.
Highlights
The interaction between protein and dsDNA is ubiquitous and plays an important role in living cells
Single-molecule fluorescence resonance energy transfer (FRET) has been successfully used to probe the structural changes of dsDNA induced by proteins as it measures accurately the change in the distance between two reporter dyes labeled on a d sDNA15–19
We studied the structural changes of dsDNA by Anabaena sensory rhodopsin transducer (ASRT) protein by using the single-molecule FRET-based cyclization assay
Summary
DNA looping at 1 M NaCl buffer solution. To observe protein-induced DNA bending with the FRET-. In the absence of ASRT, no changes in FRET histograms were observed at the same concentrations of NaCl and M gCl2, confirming that the DNA cyclization is induced by ASRT. After induction of ASRT protein at two different IPTG concentrations (0.1 and 0.8 mM), the activity of β-galactosidase was increased in all constructs with different efficiency, which implies this reporter gene expression was caused by the presence of ASRT This result is in consistent with the previous study in which the similar regulatory role of ASRT on the pec promoter-controlled reporter gene expression was observed upon IPTG induction in the presence and absence of ASR o peron[29]
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