Isotropic diffusion of the small ribosomal subunit in Escherichia coli

TL;DR

This study used advanced 3D single-particle tracking to analyze the diffusion behavior of 30S ribosomal subunits in live E. coli cells, revealing isotropic diffusion and two distinct diffusive states.

Contribution

It provides the first detailed 3D diffusion measurements of ribosomal subunits in live bacteria, highlighting isotropic diffusion and identifying two diffusive states.

Findings

30S subunits diffuse isotropically with similar coefficients in all directions.

Two diffusive states are identified, likely corresponding to translating ribosomes and free subunits.

Average diffusion coefficient is approximately 0.05 um^2/s.

Abstract

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.