Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations

Abstract

Elongation factor 4 (EF4) is one of the most conserved proteins present in bacteria as well as in mitochondria and chloroplasts of eukaryotes. Although EF4 has the unique ability to catalyze the back-translocation reaction on posttranslocation state ribosomes, the physiological role of EF4 remains unclear. Here we demonstrate that EF4 is stored at the membrane of Escherichia coli cells and released into the cytoplasm upon conditions of high ionic strength or low temperature. Under such conditions, wild-type E. coli cells overgrow mutant cells lacking the EF4 gene within 5-10 generations. Elevated intracellular Mg(2+) concentrations or low temperature retard bacterial growth and inhibit protein synthesis, probably because of formation of aberrant elongating ribosomal states. We suggest that EF4 binds to these stuck ribosomes and remobilizes them, consistent with the EF4-dependent enhancement (fivefold) in protein synthesis observed under these unfavorable conditions. The strong selective advantage conferred by the presence of EF4 at high intracellular ionic strength or low temperatures explains the ubiquitous distribution and high conservation of EF4.

Fig. 1.

Growth competition. Equal mixtures of E. coli wild-type cells and ΔlepA cells were grown under various conditions as indicated and the fraction of ΔlepA cells was determined (mutant cells in %), as described in the text and Materials and Methods.

Fig. 2.

EF4 distribution between cytosol and membranes. (Left) Immunoblotting using polyclonal anti-EF4 antibody as described in Materials and Methods. Total, the total amount of EF4 in a cell sample corresponding to the cell fraction used for EF4 determination in the cytosol and membranes, respectively. (Center) Estimates of the relative amounts (%) of EF4 in membrane and cytosol fractions based on the intensities of the corresponding immunoblotting bands. (Right) Molar ratio between EF4 and ribosomal protein S7 in the cytosol fraction based on intensities of corresponding immunoblotting bands (14), using polyclonal anti-EF4 and anti-S7 antibodies; band intensities were calculated as described in Materials and Methods.

Fig. 3.

EF4 effects on rate and accuracy of in vitro protein synthesis. (A, Left) Kinetics at 30 °C of poly(Phe) synthesis under optimal (4.5 mM) and high Mg²⁺ concentrations (14 mM) using S100 enzymes, which already contain EF4. (A, Right) Leu misincorporation per 1,000 incorporated Phe under the same conditions. (B, Left) Kinetics of oligo(Phe) in a pure system containing precharged 10 Phe-tRNAs per ribosome and the factors EF-Tu, EF-Ts, EF-G, and ± EF4. (B, Right) Oligo(Phe) incorporation in the pure system after 20 min at optimal and unfavorable Mg²⁺ concentrations (4.5 and 30 mM Mg²⁺, respectively). (C) Oligo(Phe) synthesis in the pure system incubated 30 °C for 60 min at various K(acetate) concentrations. (D) Oligo(Phe) synthesis in the pure system at various ionic concentrations either at 30 or 20 °C for 20 min as indicated.

Fig. 4.

Model showing EF4 importance at high ionic strength. (A) Normal protein synthesis on polysomes; (B) high ionic strength can lead to a single stalled ribosome on a polysome; (C) upstream ribosomes are therefore blocked stopping protein synthesis on this mRNA. (D) EF4 addition mobilizes and rescues the stalled ribosome by provoking a back-translocation resulting in a pretranslocational state that can be correctly translocated by EF-G and thus allows translation of the preceding ribosomes as well.