Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules

摘要

Using total intrnal reflection fluorescence microscopy, we directly visualize in real-time, the 1D Brownian motion and transcription elongation of T7 RNA polymerase along aligned DNA molecules bound to substrates by molecular combing. We fluorescently label T7 RNA polymerase with antibodies and use flow to convect them orthogonally to the DNA alignment direction, permitting observation and estimation of the protein diffusivity along the DNA at the single-molecule level. Our observations suggest that the 1D diffusion coefficient varies from molecule to molecule over the range 6.1 × 10⁻¹¹ cm²/s to 4.3 × 10⁻⁹ cm²/s. We also observe binding and transcription by T7 RNA polymerases on single combed T7 DNA molecules with an apparent association rate of 1.6 μM⁻¹s⁻¹. From the measured dependence of the rate of transcription on concentration of nucleotide triphosphate, we infer that the combed DNA molecules capable of interacting with proteins are under an average tension of 25 pN.