Molecular evolution of protein-RNA mimicry as a mechanism for translational control

Abstract

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the ribosome peptidyl transferase center and mimics the 3'-acceptor stem of tRNA. Although the EF-P acceptor loop residues required for PoxA recognition are highly conserved, their conservation was found to be independent of the phylogenetic distribution of PoxA. This suggests EF-P first evolved tRNA mimicry to optimize interactions with the ribosome, with PoxA-catalyzed aminoacylation evolving later as a secondary mechanism to further improve ribosome binding and translation control.

Figure 1.

Contact surface in EF-P/PoxA and tRNA^Asp/AspRS complexes. The image shows a comparison of the polar contacts between EF-P and PoxA with polar contacts between tRNA^Asp and AspRS. Image corresponds to a superposition of AspRS and PoxA from pdb files 1asy (chains A and R) and 3a5z (chains C and D). AspRS is in blue, tRNA^Asp in orange, PoxA in magenta and EFP in green. Amino acids and nucleotides making polar contacts are marked in balls with the following colors: AspRS in light blue, tRNA^Asp in light orange, EFP in light green and PoxA in red. (A) Enzymes. (B) Substrates.

Figure 2.

EF-P identity elements. (A) Active site of PoxA. PoxA residues required for aminoacylation of EF-P and the corresponding EF-P contact residues are highlighted. The α-lysyl-adenylate analog 5′-O-[(l-lysylamino) sulfonyl] adenosine (KAA) is also represented. (B) tRNA structural mimicry by EF-P identity elements. EF-P identity elements and the relevant tRNA nucleotides are shown as localized after superposition of PoxA and AspRS. (C) Active site of PoxA. PoxA amino acids from the medium effect group and the EF-P amino acids contacted by them are highlighted. Colors used in this figure correspond to the color scheme of Figure 1.

Figure 3.

Replacement of F29, K31 or G33 with alanine prevents EF-P recognition by PoxA. WT and mutant EF-P were aminoacylated with PoxA using ¹⁴C-α-lysine. (A) Plot shows aminoacylation of WT (circle), F29A (square), K31A (rhombus) and G33A (triangle) EF-P with ¹⁴C-α-Lys. (B) After 6 h, aminoacylation samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Top: coomassie stained gel; bottom: phosphor image of the gel.

Figure 4.

Conservation of EF-P residues involved in PoxA contacts. WebLogo representation of a fragment of the alignment of diverse EF-Ps that contain a lysine in the equivalent to the modification position. The top WebLogo (A) corresponds to an alignment of 14 EF-P sequences from organisms that have a poxA gene encoded in their genome. The bottom WebLogo (B) corresponds to an alignment of 17 EF-P sequences from organisms that do not have a poxA gene. The acceptor loop is marked with a black line, with the modification position highlighted with a triangle. Contacts to PoxA (based on pdb 3a5z) are marked with red triangles and contacts to the ribosome (pdb 3huw and 3hux) are indicated with cyan triangles. Numbering of the WebLogo positions corresponds to the full alignment, and corresponding positions on EF-P from E. coli are indicated below the triangles (for full alignments see Supplementary Figure S4).

Figure 5.

EF-P contacts with PoxA and the ribosome. Figure shows EF-P structures from pdb 3a5a-D (A and C) or 3huw-V (B and D). Atoms from PoxA (C and E) or the ribosome and tRNA (D and F) that contact EF-P are highlighted in red. Variable positions of the alignments (Figure 4 and Supplementary Figure S4) are highlighted on the EF-P surface for organisms with (C and D) or without (E and F) poxA. Variable positions are in green and non-variable are in blue.

Figure 6.

Complementation of Salmonella ΔpoxA with poxA variants. Figure shows complementation assays for phenotypes associated with poxA deletion mutants. Sensitivity to low osmolarity was assessed by the size of colonies grown on solid AB2 media (A), whereas sensitivity to gentamicin (3.125 µg/ml) (B) and lauryl sulfobetaine (6.25 mM) (C) was assessed as turbidity of cells grown on liquid media in presence of the corresponding compound.