Kim Ji Hoon, Larson Ronald G
Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109-2136, USA.
Nucleic Acids Res. 2007;35(11):3848-58. doi: 10.1093/nar/gkm332. Epub 2007 May 25.
Using total internal 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 x 10(-11) cm2/s to 4.3 x 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 microM(-1)s(-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.
利用全内反射荧光显微镜，我们实时直接观察到了T7 RNA聚合酶沿着通过分子梳结合到底物上的排列好的DNA分子的一维布朗运动和转录延伸。我们用抗体对T7 RNA聚合酶进行荧光标记，并利用流动使它们与DNA排列方向正交对流，从而在单分子水平上观察和估计蛋白质沿DNA的扩散率。我们的观察结果表明，一维扩散系数在分子间变化，范围为6.1×10⁻¹¹平方厘米/秒至4.3×10⁻⁹平方厘米/秒。我们还观察到T7 RNA聚合酶在单个梳理的T7 DNA分子上的结合和转录，其表观缔合速率为1.6微摩尔⁻¹秒⁻¹。从测量的转录速率对三磷酸核苷酸浓度的依赖性，我们推断能够与蛋白质相互作用的梳理DNA分子平均承受25皮牛的张力。
