Single Molecule Tracking of Elongation Factor Tu (EF-Tu) in Live E. Coli

Abstract

Translation requires efficient peptidyl transfer to support the cellular needs. Of the factors that facilitate the peptide elongation, elongation factor Tu (EF-Tu) is crucial in helping the correct aminoacyl-tRNA to be incorporated into the protein. It also acts as an energy carrier to move aminoacyl-tRNA near to P site peptides, forming a new peptide bond. Here, we investigated diffusive motions of translationally fused EF-Tu (TufA and TufB) with mEos2 via single particle tracking photoactivation localization microscopy (spt-PALM) on live E. coli cells. The mean diffusion coefficient of EF-Tu tracked at 100 Hz was 0.15 um∧2/s. The spatial distribution of EF-Tu is reminiscent of that of translating ribosomes and its diffusion coefficient is five times faster than 70S diffusion (0.03 um∧2/s). However, this is still much slower compared to the expected diffusion of free EF-Tu-size proteins such as GFP (5 um∧2/s). This suggests that EF-Tu spends most of the time on ribosome or/and on a large complex. We will further investigate the cause of slow diffusive motion via antibiotic treatments and mutations on translation machinery.