Isotropic diffusion of the small ribosomal subunit in Escherichia coli

The ribosome is one of the most important macromolecular complexes in a living cell. We have tracked individual 30S ribosomal subunits in exponentially growing E. coli cells using three-dimensional single-particle tracking, where the z-position is estimated using astigmatism. The 30S subunits are stochiometrically labeled with S2-mEos2 by replacing the rpsB gene in the E. coli chromosome by rpsB-mEos2. The spatial precision in tracking is 20 nm in xy and 70 nm in z. The average trajectory consists of 4 steps corresponding to 80 ms. The trajectories are excluded from parts of the cell, consistent with nucleoid exclusion, and display isotropic diffusion with nearly identical apparent diffusion coefficients (0.05 +/- 0.01 um^2/s) in x, y and z. The tracking data fits well to a two-state diffusion model where 46% of the molecules are diffusing at 0.02 um^2/s and 54% are diffusing at 0.14 um^2/s. These states could correspond to translating 70S ribosomes and free 30S subunits.