Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms

•Methods for elucidation of transcription mechanisms by single-molecule fluorescence.•Processing of multi-wavelength single-molecule co-localization data.•Analysis of single-molecule binding and dissociation kinetics. Multi-wavelength single molecule fluorescence microscopy is a valuable tool for cl...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2015-09, Vol.86, p.27-36
Main Authors: Friedman, Larry J., Gelles, Jeff
Format: Article
Language:English
Subjects:
DNA - chemistry
DNA-Directed RNA Polymerases - chemistry
DNA-Directed RNA Polymerases - genetics
Fluorescence microscopy
Kinetics
Microscopy, Fluorescence
Models, Theoretical
Nanotechnology - methods
Photobleaching
Promoter Regions, Genetic
Single molecule statistics
TIRF
Transcription regulation
Transcription, Genetic
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Summary:•Methods for elucidation of transcription mechanisms by single-molecule fluorescence.•Processing of multi-wavelength single-molecule co-localization data.•Analysis of single-molecule binding and dissociation kinetics. 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.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2015.05.026