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. 2014 Oct 23;9(2):476-83.
doi: 10.1016/j.celrep.2014.09.008. Epub 2014 Oct 9.

A Conserved Proline Triplet in Val-tRNA Synthetase and the Origin of Elongation Factor P

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Free PMC article

A Conserved Proline Triplet in Val-tRNA Synthetase and the Origin of Elongation Factor P

Agata L Starosta et al. Cell Rep. .
Free PMC article

Abstract

Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNA(Val) with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNA(Val) with valine and preventing formation of mischarged Thr-tRNA(Val) as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.

Figures

Fig. 1
Fig. 1. A conserved proline triplet in ValS confers EF-P dependence for expression
(A) Scatter-plot illustrating the total number of polyproline-containing proteins relative to genome size [in terms of open reading frame (ORF) number] of species within different bacterial phyla. (B) Zoom of (A) highlighting bacteria with the smallest genomes. (C) Sequence alignment illustrating the conservation of PPP and GPP in Val- and Ile-tRNA synthetases, respectively, across the three domains of life, compared with the highly conserved “HIGH” motif. (D) Heat map representation of changes in expression levels in vivo (blue, down-regulated; red, up-regulated; white, unchanged; grey, not identified or quantified) of selected proteins when comparing Δefp, ΔyjeA, ΔyjeK and ΔyfcM strains against a wildtype E. coli internal standard. Data from two biological replicates taken from (Peil et al., 2013). (E) Schematic illustration of the gene arrangement of the ilvL-BN and ilvL-GMEDA operons, with the corresponding sequence of the leader peptides IvbL and IlvL. Valine residues essential for attenuation are shown in red. (F) Autoradiograph of SDS-PAGE monitoring the in vitro translation at 3 min and 7 min of ValS, in the absence (-EF-P) and presence of EF-P (+EF-P), with full-length (FL) ValS and peptidyl-tRNA (25 kDa) as indicated. (G) Toe-printing of ValS in the absence (-) and presence (+) of active EF-P. G and C sequence lanes are included.
Fig. 2
Fig. 2. PPP at the active site of ValS is required for efficient tRNA charging
(A) Location of PPP in the active site of ValS relative to Val-AMP (PDB1GAX) (Fukai et al., 2000). (B) Active site of IleS with Ile-AMP (1JZQ) (Nakama et al., 2001). (C) ValS from (A) but with superimposed position of Ile-AMP from (B). (D) as in (C) but with in silico Pro41Gly mutation. (E) Charging efficiency of tRNAVal with valine by wildtype (wt) ValS and ValS mutants as a function of time (min). (F,G) Autoradiograph of TLC separation of (F) γ[32P]-Pi from γ[32P]-ATP, and (G) α[32P]-AMP and α[32P]-ADP from α[32P]-ATP, when wildtype ValS (wt), or ValS mutant(s) were incubated with valine, in the absence (-) and presence (+) of pyrophosphatase (PPi-ase) and/or deacylated tRNAVal. ATP at the origin indicates where the samples were loaded onto the TLC plate. In (F), the migration position of Pi is determined by treatment of γ[32P]-ATP with Apyrase (A), and in (G), the migration of AMP and ADP was determined by treatment of α[32P]-ATP with increasing concentrations of Apyrase.
Fig. 3
Fig. 3. PPP at the active site of ValS is required to prevent Thr-tRNAVal formation.
(A) Scheme for misactivation and mischarging of tRNAVal with threonine and subsequent deacylation by ValS. (B) Overview of ValS bound with tRNA (yellow) and Thr-AMP (orange) (PDB1GAX) (Fukai et al., 2000). PPP and T222 are indicated by red spheres. Inset shows the active site of ValS with PPP relative to Thr-AMP. (C) Mischarging of tRNAVal with threonine by wildtype (wt) ValS and ValS mutants as a function of time (min). (D) Autoradiograph of TLC separation of α[32P]-AMP and α[32P]-ADP from α[32P]-ATP, when wildtype ValS (wt), or ValS mutant(s) were incubated with threonine, in the absence (-) and presence (+) of deacylated tRNAVal. ATP at the origin indicates where the samples were loaded onto the TLC plate and apyrase treatment of ATP provides markers for ADP and AMP positions.
Fig. 4
Fig. 4. The proline triplet of ValS is required for viability of E. coli.
(A) Scheme for ValS complementation system. (B-G) Growth curves (OD600nm) of E. coli strains (B) BW25113, JL001, JL001 + pUlp1, or (C-G) JL001 bearing additional plasmids expressing pUlp1, and/or (C) wildtype ValS (pValS-PPP) or ValS mutants (D) pValS-GPP, (E) pValS-PGP, (F) pValS-PPG or (G) pValS-GGG. (H) Comparison of growth (after 90 min) of JL001 bearing plasmids expressing pUlp1 and pValS/XPP mutants normalized by subtraction of background growth in the presence of pUlp1 alone.

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