Single-molecule imaging for investigating the transcriptional control

Transcription is an essential biological process involving numerous factors, including transcription factors (TFs), which play a central role in this process by binding to their cognate DNA motifs. Although cells must tightly regulate the kinetics of factor association and dissociation during transcription, factor dynamics during transcription remain poorly characterized, primarily because of the reliance on ensemble experiments that average out molecular heterogeneity. Recent advances in single-molecule fluorescence imaging techniques have enabled the exploration of TF dynamics at unprecedented resolution. Findings on the temporal dynamics of individual TFs have challenged classical models and provided new insights into transcriptional regulation. Single-molecule imaging has also elucidated the assembly kinetics of transcription complexes. In this review, we describe the single-molecule fluorescence imaging methods widely used to determine factor dynamics during transcription. We highlight new findings on TF binding to chromatin, TF target search, and the assembly order of transcription complexes. Additionally, we discuss the remaining challenges in achieving a comprehensive understanding of the temporal regulation of transcription.