Probing intracellular kinetics at the level of single molecules
by
Johan Elf(Uppsala)
→
Europe/Stockholm
122:028
122:028
Description
I will present our resent advancements in tracking individual freely diffusing fluorescent proteins molecules at high time resolution in the cytoplasm of bacterial cells. In vivo tracking of individual proteins molecules makes it possible to study kinetics high time resolution without synchronizing the population of molecules. For example by monitoring the kinetics of the response mediator RelA we have developed a single molecule assay to study stress response and starvation at the level of individual bacteria.
The RelA protein binds to a small fraction of ribosomes, where it synthesizes the global transcriptional regulator ppGpp in response to amino acids deprivation. This the ppGpp molecule binds to the RNAP and rapidly reprograms the cell for the new environment, in what is called the stringent response. While E. coli contains on average about 100 RelA molecules, using a photo-activatable fluorescent probe we can activate only a few fluorescent molecules per cell at any given time and track them at high time resolution. The procedure can be repeated many times to get accurate statistics for in individual cells.
When the cell grows exponentially, RelA trajectories closely resemble trajectories of fluorescently tagged ribosomal proteins (D~0.4um^2/sec as compared to D~0.3 um^2/sec for ribosomes). After nutritional downshift, RelA binding kinetics changes rapidly and the protein diffuses very fast (D~3.5um^2/sec) as if it only binds to ribosomes transiently. The assay has made it possible to study the rapid and transient stringent response in individual cell as well as the heterogeneity in the stress response over the population.
