Probing transcription factor dynamics at the single-molecule level in a living cell

Science. 2007 May 25;316(5828):1191-4. doi: 10.1126/science.1141967.

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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • DNA, Bacterial / metabolism*
  • Diffusion
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / metabolism*
  • Kinetics
  • Lac Operon*
  • Lac Repressors
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Operator Regions, Genetic
  • Protein Binding
  • Repressor Proteins / metabolism*
  • Signal Transduction

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Lac Repressors
  • LacI protein, E coli
  • Luminescent Proteins
  • Repressor Proteins
  • yellow fluorescent protein, Bacteria