Single-molecule nanomanipulation of supercoiled DNA permits measurement, in real time, of spatial and temporal parameters of protein-DNA interactions that affect DNA topology1-7. In this method, a double-stranded DNA molecule containing at least one target for the protein of interest is attached at one end to a magnetic bead and at the other end to a glass surface. The experimental setup and the monitoring of the end-to-end extension (l) of the stretched, supercoiled DNA molecule is diagramed in Figure 1a. The protein of interest is introduced into the system, and protein-dependent changes in DNA linking number (Lk) or DNA twist (Tw) are detected as changes in the number of plectonemic supercoils (changes in DNA writhe, Wr; Lk = Tw + Wr; ref. 8) and corresponding changes in l (Fig. 1b-d). This approach has been applied to analysis of supercoil formation and relaxation by topoisomerases1-5 and to promoter unwinding by bacterial RNA polymerase (RNAP)6,7. The spatial and temporal resolution of the method is expected to increase with decreasing length of the supercoiled DNA segment (equations in ref. 6). (Reducing the length of the supercoilable DNA segment should have no effect on the amplitude of changes in DNA extension resulting from protein-dependent changes in DNA topology (signal), but should reduce the amplitude of random fluctuations in DNA extension (noise), thereby resulting in improvement in the signal-to-noise ratio.) Previous work has involved supercoiled DNA segments 4-44 kilobases (kb) in length1-7. Here, we describe preparation of DNA molecules with 2-kb supercoilable DNA segments and document superior resolution in analysis of promoter unwinding and DNA compaction by bacterial RNAP.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology