Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites - PubMed (original) (raw)

Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites

Li Li et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Mycoplasma parasites escape host immune responses via mechanisms that depend on remarkable phenotypic plasticity. Identification of these mechanisms is of great current interest. The aminoacyl-tRNA synthetases (AARSs) attach amino acids to their cognate tRNAs, but occasionally make errors that substitute closely similar amino acids. AARS editing pathways clear errors to avoid mistranslation during protein synthesis. We show here that AARSs in Mycoplasma parasites have point mutations and deletions in their respective editing domains. The deleterious effect on editing was confirmed with a specific example studied in vitro. In vivo mistranslation was determined by mass spectrometric analysis of proteins produced in the parasite. These mistranslations are uniform cases where the predicted closely similar amino acid replaced the correct one. Thus, natural AARS editing-domain mutations in Mycoplasma parasites cause mistranslation. We raise the possibility that these mutations evolved as a mechanism for antigen diversity to escape host defense systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Degenerated editing domains of Mycoplasma AARS. (A) Phylogenetic tree of Mycoplasma based on 16S rRNA. Bootstrap values are shown for each node and scale bar denotes substitutions per site. Predicted editing-defective AARS are indicated in boxes (Right) (see also

Figs. S1–S3

). (B) Alignment of LeuRS CP1 domain with key editing site residues indicated (see also

Table S3

). Shaded and black boxes represent conserved and homologous residues. (C) Tandem MS analysis of precursor peak m/z = 796.4 at z = 3 identified a mistranslated peptide in M. mobile. A fragment peak (m/z = 411.2; _y_3 ion of YPIILEDGFSEHDWDA(Y)TK; phosphopyruvate hydratase) was confirmed by the balance of the fragmentation spectrum. (Inset) Predicted _y_2 and _y_3 ion positions for the genome-encoded peptide (dotted bars) and observed mistranslated product (solid bars; F → Y). (D) Identification of faithfully translated peptide YPIILEDGFSEHDWDA(F)TK for phosphopyruvate hydratase (peak of m/z = 395.2 at the _y_3 ion position).

Fig. 2.

Tandem MS analysis of single peptides from M. mobile and E. coli that express M. mobile LeuRS demonstrate mistranslation of leucine codons. An L → V substitution of M. mobile peptides LTKLINS(L/V)K from phosphopentomutase (A) and IKTSVKN(L/V)AK from phosphotransacetylase (B) respectively generated peaks of m/z = 869.2 [confirmatory _b_8 ion of LTKLINS(V)K; precursor peak m/z = 508.5 at z = 2] and m/z = 870.2 [confirmatory _b_8 ion of IKTSVKN(V)AK; precursor peak m/z = 544.3 at z = 2]. (C) An L → V substitution of E. coli peptide LINQGMI(L)GK generated a peak of m/z = 303.2 corresponding to the confirmatory _y_3 ion of LINQGMI(V)GK. An L → M substitution of E. coli peptides HLENKIIKKEIYIAKKI(L)NFII (D) and SKDKFYA(L)D MFPYPSGSGLHVGHPEGYTATDIISR (E) respectively generated peaks of m/z = 1105.7 [confirmatory formula image ion of HLENKIIKKEIY IAKKI(Mox)NFII] and m/z = 953.5 [confirmatory _b_8 ion of SKDKFYA(Mox)DMFPYPSGSGLHVGHPEGYTATDIISR]. Methionine was typically oxidized to methionine sulfoxide (see also

Fig. S4

Fig. 3.

Wild-type M. mobile LeuRS mischarges tRNALeu. Deacylation reactions contained approximately 20 μM [3H]-Val-tRNALeu (A), approximately 6.5 μM [3H]-Ile-tRNALeu (B) or 100 μM [35S]-Met-tRNALeu (C) and 100 nM M. mobile, E. coli wild-type or mutant LeuRS. Mischarging assays incorporated 160 μM [14C]-valine (50 μCi/mL; D), 21 μM [3H]-isoleucine (166 Ci/mmol; E) or 20 μM [35S]-methionine (20 μCi/mL; F) and 1 μM M. mobile or E. coli LeuRS. Editing-defective E. coli LeuRS (Ec-Ed) mutant is a positive control. (G) Acid gel of tRNALeu charged with [35S]-methionine. Abbreviations: (square), Ec-WT; (inverted triangle), Mm; (triangle), Ec-Ed; and (diamond), no enzyme control (No E). Error bars represent standard deviations from triplicated reactions.

Fig. 4.

Proposed evolutionary scheme for LeuRS CP1 editing module degeneration in Mycoplasma. M. synoviae and M. agalactiae LeuRS were acquired from Proteobacteria via horizontal gene transfer (

Fig. S8

). Primary structure degeneration of the CP1 domain resulted in M. agalactiae and M. synoviae LeuRS CP1a and CP1s (Left). Genome reduction in M. mobile completely eliminated the CP1 editing module in LeuRS (ΔCP1; Right).

References

1. Maniloff J. In: Molecular Biology and Pathogenicity of Mycoplasmas. Razin S, Herrmann R, editors. New York: Kluwer Academic; 2002. pp. 31–44.
1. Rottem S. Interaction of Mycoplasmas with host cells. Physiol Rev. 2003;83:417–432. - PubMed
1. Ibba M, Söll D. Quality control mechanisms during translation. Science. 1999;286:1893–1897. - PubMed
1. Fersht AR. Sieves in sequence. Science. 1998;280:541. - PubMed
1. An S, Musier-Forsyth K. Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK protein. J Biol Chem. 2004;279:42359–42362. - PubMed

Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites - PubMed (original) (raw)