Quantifying the Binding and Target-Search Kinetics of Transcriptional Regulatory Factors by Live-Cell Single-Molecule...

Eukaryotic transcriptional regulatory factors, such as transcription factors and epigenetic regulatory factors, must locate, bind, and assemble at specific genomic regions to execute functions within the complex and crowded environment of the nucleus. These dynamic processes are typically at nonequilibrium, so quantifying their binding and target-search processes within the native environment is essential for understanding transcriptional mechanisms. Live-cell single-molecule tracking (SMT) is an emerging technique that can be utilized to observe molecular trajectories of individual transcriptional regulatory complexes within the nucleus. Here, we describe the use of live-cell SMT to observe trajectories of individual transcriptional regulatory complexes. We delineate the imaging analysis to obtain chromatin-bound fraction and residence time. Finally, we elaborate on the kinetic modeling to estimate target-search parameters. These binding and target-search parameters facilitate the understanding of how transcription is spatially and temporally regulated under physiological and pathological conditions.