Spatial Distribution and Diffusive Motion of RNA Polymerase in Live Escherichia coli
Spatial Distribution and Diffusive Motion of RNA Polymerase in Live Escherichia coli
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DOI：
10.1128/jb.00198-11
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发表时间：
2011-10-01
影响因子：
3.2
通讯作者：
Weisshaar, James C.
中科院分区：
文献类型：
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作者：
Bratton, Benjamin P.;Mooney, Rachel A.;Weisshaar, James C.
通过标记RNA聚合酶β '亚基（RNAP），首次用荧光显微镜研究了RNAP在大肠杆菌活细胞中的空间分布和扩散运动。在40 ms的时间分辨率下，空间分布表现出两个或三个窄峰，半峰全宽为300- 600 nm，在1 s内保持其位置在60 nm以内。这些特征的强度占总强度的20%至30%。光漂白后的荧光恢复（FRAP）测量RNAP在1 μ m长度尺度上的扩散运动。在许多细胞上平均，53% +/- 19%的RNAP分子在3秒时间尺度上是移动的，平均表观扩散常数< D（RNAP）>为0.22 +/- 0.16 μ m（2）-s（-1）。剩下的47%甚至在30秒的时间尺度上都是静止的。我们解释不动的部分所产生的RNAP特异性结合到DNA，积极转录或不。移动的部分（f（移动的））的扩散运动可能涉及与DNA非特异性结合期间的一维滑动和DNA链之间的三维跳跃。在D（RNAP）和f（移动的）中存在显著的细胞间异质性。
By labeling the beta' subunit of RNA polymerase (RNAP), we used fluorescence microscopy to study the spatial distribution and diffusive motion of RNAP in live Escherichia coli cells for the first time. With a 40-ms time resolution, the spatial distribution exhibits two or three narrow peaks of 300-to 600-nm full width at half-maximum that maintain their positions within 60 nm over 1 s. The intensity in these features is 20 to 30% of the total. Fluorescence recovery after photobleaching (FRAP) measures the diffusive motion of RNAP on the 1-mu m length scale. Averaged over many cells, 53% +/- 19% of the RNAP molecules were mobile on the 3-s timescale, with a mean apparent diffusion constant < D(RNAP)> of 0.22 +/- 0.16 mu m(2)-s(-1). The remaining 47% were immobile even on the 30-s timescale. We interpret the immobile fraction as arising from RNAP specifically bound to DNA, either actively transcribing or not. The diffusive motion of the mobile fraction (f(mobile)) probably involves both one-dimensional sliding during nonspecific binding to DNA and three-dimensional hopping between DNA strands. There is significant cell-to-cell heterogeneity in both D(RNAP) and f(mobile).