| Summary: | Previous studies have shown that translocation of an actively transcribing RNA polymerase leads to local increases or decreases in DNA torsion (twin-supercoiled domain), which cannot be resolved in vivo due to interactions of the template DNA, nascent RNA and polymerase with the crowded cellular environment. Local changes in DNA supercoiling are biologically relevant as they have been shown to regulate transcription initiation at promoters located downstream. Current in vitro single-molecule approaches are not able to directly probe transcription-coupled DNA supercoiling due to an inability to simultaneously monitor changes in torsional stress and localise individual transcribing RNA polymerase(s) on the DNA. Described here is a novel optical microscope, which utilises a combination of magnetic tweezers, bright-field illumination and wide-field epifluorescence imaging to permit the visualisation of fluorescently tagged polymerases transcribing in real-time on a torsionally constrained DNA template. With this unique geometry, transcription as a function of applied torsion can be probed directly in vitro. Unlike standard magnetic tweezers configurations this system extends tethers horizontally relative to the microscope slide surface, which allows individual enzymes to be directly viewed via attached fluorophores. The magnetic tweezers allow both the relative extension and linking number of the DNA tether to be manipulated, thus enabling transcription to be studied under conditions of constant DNA extension and defined torsional stress.
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