Interactions of elongation factor EF‐P with the Escherichia coli ribosome

Abstract

EF‐P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF‐P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T‐site). EF‐P also protects domain V of the 23S rRNA proximal to the A‐site (50S T‐site) and more strongly the A‐site of 70S ribosomes. We suggest that EF‐P: (a) may play a role in translational fidelity and (b) prevents entry of fMet–tRNA into the A‐site enabling it to bind to the 50S P‐site. We also report that EF‐P promotes a ribosome‐dependent accommodation of fMet–tRNA into the 70S P‐site.

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

“…4 The crystal structure of the T. thermophilus 70 S ribosome complex with EF-P, a short mRNA, and the initiator formylMet-tRNAi revealed that EF-P associates with ribosomal protein L1 and that in the presence of tRNAi, EF-P binds between the peptidyl-tRNA site and the exit tRNA site of the ribosome (7), with the basic side chain of Arg-32 directed toward the peptidyltransferase center. The interaction of EF-P with the L1 50 S ribosomal protein is consistent with its proposed action in translocation because the protein promotes the ribosomal accommodation of formyl-Met-tRNA from the aminoacyl-tRNA site to the peptidyl-tRNA site (23). It was proposed that EF-P facilitates the proper positioning of the initiator tRNA for the formation of the first peptide bond.…”

“…In view of the proposed role for EF-P as a factor involved in stress adaptation in Salmonella (27), it is interesting to note the genetic connection between several eIF5A genes that enhance tolerance to heat, osmotic, oxidative, and nutrient stresses in plants (41,42). Under such a scenario, the exact mechanism used by EF-P and eIF5A to selectively stimulate the translocation of specific mRNAs and the nature of such mRNAs (3,23,43,44) warrant future investigations.…”

“…Although the deoxyhypusine/hypusine modification site is a strictly conserved lysine in aIF5A and eIF5A, conservation is relaxed in EF-P to allow Lys, Arg, Asn, or Gln (22). Interestingly, mass spectrometric analyses of the tryptic peptides of Escherichia coli native EF-P suggested a novel post-translational modification of EF-P (different from the hypusine modification) by the addition of a mass of 144 Da at Lys-34 (23), the site corresponding to the hypusine modification site of eIF5A.…”

The hypusine-containing translation factor eIF5A

“…However, several studies demonstrated that EF-P, at least in some bacteria, is modified. Mass spectrometry analysis of tryptic peptides from native EF-P purified from E. coli revealed that Lys34 was modified, and that the modification contributed an extra mass of ~144 Da (Aoki et al, 2008). Based on comparative genomics analyses including gene clustering and phylogenetic conservation it was hypothesized that the E. coli genes yjeA and yjeK , encoding a truncated lysyl-tRNA synthetase-related protein and a protein related to lysine aminomutase, respectively, function in a pathway to modify EF-P (Bailly and de Crecy-Lagard, 2010).…”

“…Biochemical studies demonstrated that YjeA/PoxA could lysinylate (lysylate) EF-P in vitro (Navarre et al, 2010; Yanagisawa et al, 2010), and this activity was dependent on the conserved Lys34 residue (which corresponds to the site of hypusine modification in eIF5A) (Navarre et al, 2010). Notably, Lys34 was also reported to be modified on native EF-P from E. coli (Aoki et al, 2008; Peil et al, 2012; Roy et al, 2011). In vitro and co-expression studies revealed that YjeA/PoxA could add a Lys residue to EF-P, increasing the mass by ~128 Da (Navarre et al, 2010; Park et al, 2012; Yanagisawa et al, 2010).…”