Mechanism of tRNA-dependent editing in translational quality control - PubMed (original) (raw)

Mechanism of tRNA-dependent editing in translational quality control

Jiqiang Ling et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNA(Phe). Deprotonation of a water molecule by the highly conserved residue betaHis-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10-fold. In contrast, substitution of the 3'-OH group of tRNA(Phe) severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

T. thermophilus PheRS editing site complexed with Tyr; equivalent residues in E. coli PheRS are shown in parentheses (adapted from ref. 21).

Fig. 2.

Fig. 2.

Tyr-tRNAPhe synthesis by PheRS variants (100 nM). □, wild-type β-subunit; ▴, βG318W; ▵, βE334A; ○, βH265A; ×, βR244A; ●, βR244A/βH265A; ▿, βT354V. Plots represent the average of three independent experiments.

Fig. 3.

Fig. 3.

Structural modeling of the PheRS editing site complexed with Tyr-A76.

Fig. 4.

Fig. 4.

Impact of PheRS βT354 replacements on ATP consumption. □, wild-type β-subunit; ○, βT354V; ×, βT354C; ▵, βT354S.

Fig. 5.

Fig. 5.

Mechanism of posttransfer editing by PheRS. (A) Kinetic scheme for PheRS editing. (B) Model for Tyr-tRNAPhe hydrolysis at the PheRS editing site (T. thermophilus numbering; see Fig. 3).

Fig. 6.

Fig. 6.

Editing of PheRS variants against cognate Phe. (A) ATP consumption in the presence of Phe (10 mM), tRNAPhe (10 μM), and PheRS (1 μM). (B) Phe-tRNAPhe hydrolysis by PheRS variants (0.5 μM). (C) Phe-tRNAPhe synthesis by PheRS variants (2 nM). ○, no enzyme; □, wild-type β-subunit; ▵, βE334A; ▴, βE334I; ×, βP263A/βY360A.

Fig. 7.

Fig. 7.

The role of the 3′-OH of A76 in editing. (A and B) Tyrosylation of A76 tRNAPhe (squares), 3′-dA76 tRNAPhe (triangles), and 3′-F-A76 tRNAPhe (circles) by 1 μM wild-type PheRS (A) or 1 μM βG318W PheRS (B). (C) Deacylation of 0.2–0.4 μM tyrosylated A76 tRNAPhe, 3′-dA76 tRNAPhe, and 3′-F-A76 tRNAPhe in the presence (filled symbols) or absence (open symbols) of 0.5 μM wild-type β-subunit PheRS.

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