Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin; Molecular Biophysics Program, University of Wisconsin-Madison, Madison, Wisconsin.
Biophys J. 2013 Dec 17;105(12):2676-86. doi: 10.1016/j.bpj.2013.10.024.
Superresolution fluorescence microscopy is used to locate single copies of RNA polymerase (RNAP) in live Escherichia coli and track their diffusive motion. On a timescale of 0.1-1 s, most copies separate remarkably cleanly into two diffusive states. The "slow" RNAPs, which move indistinguishably from DNA loci, are assigned to specifically bound copies (with fractional population ftrxn) that are initiating transcription, elongating, pausing, or awaiting termination. The "mixed-state" RNAP copies, with effective diffusion constant Dmixed = 0.21 μm(2) s(-1), are assigned as a rapidly exchanging mixture of nonspecifically bound copies (fns) and copies undergoing free, three-dimensional diffusion within the nucleoids (ffree). Longer trajectories of 7-s duration reveal transitions between the slow and mixed states, corroborating the assignments. Short trajectories of 20-ms duration enable direct observation of the freely diffusing RNAP copies, yielding Dfree = 0.7 μm(2) s(-1). Analysis of single-particle trajectories provides quantitative estimates of the partitioning of RNAP into different states of activity: ftrxn = 0.54 ± 0.07, fns = 0.28 ± 0.05, ffree = 0.12 ± 0.03, and fnb = 0.06 ± 0.05 (fraction unable to bind to DNA on a 1-s timescale). These fractions disagree with earlier estimates.
超分辨率荧光显微镜被用于在活的大肠杆菌中定位单个 RNA 聚合酶 (RNAP) 分子，并追踪它们的扩散运动。在 0.1-1 秒的时间尺度内，大多数分子会明显地分离成两种扩散状态。“慢”RNAP 与 DNA 位点难以区分，被分配到特定结合的分子（具有分数种群 ftrxn），这些分子正在起始转录、延伸、暂停或等待终止。“混合态”RNAP 分子的有效扩散常数 Dmixed = 0.21 μm(2) s(-1)，被分配为非特异性结合分子（fns）和在核仁中自由进行三维扩散的分子（ffree）的快速交换混合物。持续 7 秒的较长轨迹揭示了慢态和混合态之间的转变，证实了这些分配。持续 20 毫秒的短轨迹能够直接观察到自由扩散的 RNAP 分子，得到 Dfree = 0.7 μm(2) s(-1)。对单个粒子轨迹的分析提供了 RNAP 进入不同活性状态的定量估计：ftrxn = 0.54 ± 0.07，fns = 0.28 ± 0.05，ffree = 0.12 ± 0.03，fnb = 0.06 ± 0.05（在 1 秒时间尺度上无法与 DNA 结合的分数）。这些分数与早期的估计值不一致。
