Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell

TLDR

Transcription factors regulate gene expression by binding to DNA. Using single‑molecule fluorescence in living *E. coli*, we tracked a lac repressor’s specific and nonspecific DNA interactions, measuring binding kinetics, 1‑D diffusion along DNA, and 3‑D translocation between segments.

Abstract

Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ∼90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.