Post-translational Modification by β-Lysylation Is Required for Activity of Escherichia coli Elongation Factor P (EF-P)

Abstract

Background: Bacterial elongation factor P (EF-P) was proposed to undergo unique post-translational modification by YjeA and YjeK. Results: We identified ␤-lysylation in native EF-P and show that ␤-lysyl-EF-P is the active form. Conclusion: ␤-Lysylation occurs in EF-P in vivo and is required for activity. Significance: ␤-Lysylation of EF-P may influence this factor binding to ribosome and regulate bacterial protein synthesis.

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

“…The ␤-lysylation of EF-P by the PoxA/YjeK pathway leads to stimulation of puromycin reactivity in vitro and post-transcriptional regulation of certain genes in vivo (2,8). These studies and structural investigations (4) supported the hypothesis that EF-P acts by stabilizing initiator tRNA in the ribosomal P-site and promoting formation of the first peptide bond.…”

“…The above analyses of polysome profiles, and previous investigations of puromycin reactivity (2,22), support a role for modified EF-P in translation elongation. To further analyze the effect of EF-P ␤-lysylation and hydroxylation on translation, puromycin reactivity was measured in the presence of modified and unmodified EF-P and a K34R variant that mimics a conserved replacement seen in numerous bacterial EF-Ps (26).…”

“…In a reaction analogous to tRNA aminoacylation, PoxA activates (R)-␤-lysine and subsequently transfers it to a conserved residue (Lys-34) in EF-P (9). Attachment of a ␤-lysyl moiety is necessary for EF-P functionality both in vivo and in vitro (2,8). Recent mass spectroscopy analyses identified a second posttranslational modification of EF-P involving hydroxylation of the C4(␥) or C5(␦) of ␤-lysyl-Lys-34 by YfcM (10).…”

“…Further evidence for a role in translation elongation comes from the ability of eIF5A to promote in vitro di-and tripeptide synthesis (12). To date, the effects of EF-P and its ␤-lysine modification on translation have largely been investigated using a puromycin reactivity assay, which does not accurately represent a physiologically relevant peptide synthesis reaction (2,13,14). We now show that EF-P functions in translation elongation, with activity dependent on ␤-lysylation, whereas the additional hydroxyl modification is dispensable.…”

Elongation Factor P and Modifying Enzyme PoxA Are Necessary for Virulence of Shigella flexneri

“…Park et al (45) showed that all of the EF-P purified from E. coli cells possesses the ␤-lysine modification, leading to the hypothesis that this modification is necessary or highly advantageous for the function of EF-P. Compared to the efp mutant, the poxA mutant of S. flexneri displayed an intermediate reduction in virulence, as well as a higher growth rate and less severe decreases in many of the affected proteins identified by proteomic analysis.…”