Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms
Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms
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DOI：
10.1016/j.ymeth.2015.05.026
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发表时间：
2015-09-15
期刊：
影响因子：
4.8
通讯作者：
Gelles, Jeff
中科院分区：
文献类型：
--
作者：
Friedman, Larry J.;Gelles, Jeff
多波长单分子荧光显微镜是一个有价值的工具，阐明转录机制，其中涉及多个组件和中间体。在这里，我们描述的分析和解释这样的单分子数据的方法。所描述的方法包括用于图像对准、漂移校正、斑点辨别的方法，以及用于分析单分子结合和解离动力学的稳健方法，所述单分子结合和解离动力学考虑非特异性结合和光漂白。最后，我们给出了一个例子，使用所得到的数据提取的动力学机制的启动子结合的细菌RNA聚合酶全酶。(C)2015 Elsevier Inc. All rights reserved.
Multi-wavelength single molecule fluorescence microscopy is a valuable tool for clarifying transcription mechanisms, which involve multiple components and intermediates. Here we describe methods for the analysis and interpretation of such single molecule data. The methods described include those for image alignment, drift correction, spot discrimination, as well as robust methods for analyzing single-molecule binding and dissociation kinetics that account for non-specific binding and photobleaching. Finally, we give an example of the use of the resulting data to extract the kinetic mechanism of promoter binding by a bacterial RNA polymerase holoenzyme. (C) 2015 Elsevier Inc. All rights reserved.