(R)-β-lysine-modified elongation factor P functions in translation elongation

Abstract

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational β-lysylation but not hydroxylation.

FIGURE 1.

Deletion of efp results in increased retention of polysomes. Shown is the quantification of representative A₂₅₄ profiles of ribosomal fractions isolated from BW25113 (WT) or Δefp, ΔpoxA, or ΔyfcM E. coli strains and separated via sucrose gradient centrifugation. Cultures were grown to mid-log and in some cases (+) exposed to 100 μg/ml chloramphenicol (CAM) for 2 min prior to harvesting. The P/M ratio for each fractionation is calculated from three separate experiments.

FIGURE 2.

Initiation of translation of poxB mRNA in the presence of β-Lys-EF-P. Representative graphs of 50 S and 30 S preinitiation complex joining are shown as light scattering over time. Rates were measured in the presence or absence of unmodified EF-P or β-Lys-EF-P and fit to the single exponential equation (y = m1 × exp(−m0 × m2) + m3 + m4 × m0). k_obs = 0.0088 ± 0.0013 s-1 (BSA), 0.0089 ± 0.0009 s⁻¹ (EF-P), and 0.0082 ± 0.0027 s⁻¹ (βLys-EF-P).

FIGURE 3.

[³⁵S]fMet-puromycin formation by the ribosome in the presence of β-Lys-EF-P and hydroxyl-β-Lys-EF-P. Puromycin reactions were performed under pseudo-first order conditions using 70SIC preincubated with variants of EF-P or BSA. Error bars represent the S.D. of three separate experiments.

FIGURE 4.

fMet-Lys dipeptide synthesis in the presence of β-Lys-EF-P. Dipeptide formation assays were performed under pseudo-first order conditions using 70SIC preincubated with variants of EF-P or BSA. Error bars represent the S.D. of three separate experiments.

FIGURE 5.

Elongation of a poly-Phe peptide in the presence of modified EF-P. The rate of poly(U)-directed poly-Phe synthesis by 70 S ribosomes was measured by the incorporation of [¹⁴C]Phe. Reactions were performed in the presence or absence of unmodified EF-P (●), β-Lys-EF-P (▴), or BSA (■), preincubated with 70 S ribosomes. Error bars represent the S.D. of three separate experiments.

FIGURE 6.

Salmonella ΔyfcM mutants do not phenocopy ΔpoxA, ΔyjeK, and Δefp mutants. Salmonella mutants deficient in yfcM exhibit phenotypes similar to that of wild type Salmonella 14028s when grown under hypoosmolar conditions (antibiotic medium 2 agar plates (A)) or when exposed to gentamicin (B), lauryl sulfobetaine (C), or S-nitrosoglutathione (GSNO) (D). ΔyfcM mutants also do not exhibit a migration defect on soft agar plates (E) or a change in 1-N-phenylnaphthylamine accumulation (NPN) (F). Data points represent the average of three independent experiments. Error bars represent S.E. For 1-N-phenylnaphthylamine accumulation, a strain lacking the AcrAB multidrug efflux pump served as a positive control, and standard errors are shown only at 0, 2.5, 5, and 9.5 min for clarity. AU, arbitrary fluorescence units.

FIGURE 7.

Growth of ΔyfcM, Δefp and ΔpoxA E. coli strains. Growth in LB at 37 °C was monitored by A₆₀₀ over time for BW25113 (WT) (■), ΔyfcM (▴), Δefp (●), and ΔpoxA () E. coli strains. OD, optical density.