Bending of Short dsDNA Upon Binding of Anabaena Sensory Rhodopsin Transducer

Single-molecule fluorescence resonance energy transfer (smFRET) has been used to monitor the intra-molecular changes of DNA or proteins. Practically, the structure information of the macromolecule is required to precisely label the molecules with two fluorescence dyes. The structure information is, however, often not available, which limits the FRET technique from application. In this study, we applied a recently developed single-molecule cyclization assay, to monitor the structural change of double stranded DNA upon binding of Anabaena sensory rhodopsin transducer (ASRT), a novel signal transducer protein involved in photo-sensory and light harvesting system in bacteria whose DNA-bound structure is unknown. We first labeled the two ends of a linear double stranded DNA with a FRET pair, from which the distance between the two ends can be estimated after binding of ASRT. However, FRET would occur only if the two ends are in the FRET range (typically a few nanometers), which cannot be guaranteed without given the structure. To overcome this problem, we placed complementary single stranded tails at the two ends of the DNA, so that the tails may interact and bind to each other and bring the two ends in close proximity. We observed the enhanced loop formation of DNA (∼100 bp) upon binding of ASRT, which suggests that ASRT regulates gene expression by facilitating loop formation of the dsDNA around itself.