Probing transcription factor dynamics at the single-molecule level in a living cell.
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 approximately 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.
Elf
Johan
J
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Li
Gene-Wei
GW
Xie
X Sunney
XS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D007763:Lac Operon; D056968:Lac Repressors; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D008164:Luminescent Proteins; D012097:Repressor Proteins; C054562:yellow fluorescent protein, Bacteria