Millisecond exposure (left) captures single-molecule movement that otherwise appears as a blur (right).


Structural analysis, biochemistry, and theoretical models have built a picture of how individual transcription factors find, bind, and regulate their target genes. Now, for the first time, Johan Elf, Gene-Wei Li, and Sunney Xie (Harvard University, Cambridge, MA) provide moving pictures of this process in living cells.

The lac operon of Escherichia coli has been one of the most well-studied model systems of transcriptional regulation. When a lac repressor protein binds its operator sequence upstream of the operon, transcription is repressed. Upon binding of lactose metabolites (or analogues thereof, such as IPTG), the repressor dissociates from the operator, which enables transcription. Previous studies measured the kinetics of lac repressor binding and dissociation indirectly by analyzing the accumulation of gene products.

To follow directly the kinetics of individual lac repressors, the team expressed a fluorescent lac repressor fusion protein. Despite lac autorepression, wild-type E. coli cells still have ∼20 copies of the repressor monomer per cell—too many to pick out individual factors from the fluorescent blur. So Elf et al. decreased this number to just a few monomers by modifying the autorepression sequence.

Using a sensitive camera in combination with a high-intensity stroboscopic laser that allowed millisecond exposure times, the team as able to capture individual repressor molecules as they moved around the cell.

In the absence of IPTG, the repressor molecules are detected as single localized spots, indicating the repressor's specific binding to the lac operator. When IPTG was added, the repressors dissociated within seconds and diffused rapidly through the cell. Upon removal of IPTG, the repressors found their binding sites in ∼1 min.

During this search phase, repressors nonspecifically bound and scanned DNA for up to 5 ms at a time. Between scans, the repressors hopped to other DNA segments in less than 0.5 ms. These are the first quantitative measurements of how DNA binding proteins search for their target sequences in a living cell.

To follow individual molecules in larger, more complex cells, additional difficulties must be overcome, such as 3D tracking in large volumes. Nevertheless, the team plans to adapt the technology to see to what extent transcription factor kinetics can also be probed in eukaryotes.


Elf, J., et al.