Resampling and editing of mischarged tRNA prior to translation elongation

Abstract

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu*GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Figure 1. PheRS Editing Site Competes with EF-Tu for Tyr-tRNA^Phe

(A) Tyrosylation by E. coli PheRS (0.75 μM) at 2°C ± 10 μM activated E. coli EF-Tu. (B) Tyrosylation by wild-type or editing-defective (βA356W) E. coli PheRS (0.25 μM) at 37°C ± 10 μM activated E. coli EF-Tu. (C) Hydrolysis of Tyr-tRNA^Phe. WT β subunit EcPheRS (0.75 μM) and activated E. coli EF-Tu (10 μM) were mixed and preincubated at 37°C for 3 min before the addition of 1 μM Tyr-tRNA^Phe. Phe-tRNA^Phe was previously shown to be stable under these conditions (Ling et al., 2007a). Error bars correspond to the standard deviation from three independent experiments.

Figure 2. Recognition of E. coli tRNA^Phe during Aminoacylation and Editing

(A) Recognition of G34 by PheRS in the aminoacylation conformation (Goldgur et al., 1997). E. coli PheRS numbering is shown (equivalent T. thermophilus residues in parentheses). (B) Relative phenylalanylation and Tyr-tRNA^Phe deacylation activities. (C) Parameters for Phe-tRNA^Phe and Tyr-tRNA^Phe binding to βA356W PheRS. Error bars correspond to the standard deviation from three independent experiments.

Figure 3. Misacylation by LeuRS and ProRS

(A) Isoleucylation of total E. coli tRNAs by wild-type and editing-defective (T252Y) E. coli LeuRS variants at 2°C (8 μM enzyme) or 37°C (2.4 μM enzyme, inset) ± 10 μM activated E. coli EF-Tu. (B) Hydrolysis of 0.5 μM Ala-tRNA^Pro at 37°C ± EF-Tu, 2.5 μM EcProRS, and 10 μM activated E. coli EF-Tu. Pro-tRNA^Pro was previously shown to be stable under these conditions (Beuning and Musier-Forsyth, 2000). Error bars correspond to the standard deviation from three independent experiments.

Figure 4. Resampling and Editing of Misacylated tRNA

(A) Poly(U)-directed poly-Phe and poly-Tyr synthesis at 37°C. As controls, 1 μM [¹⁴C] Phe or [³H] Tyr were added instead of [¹⁴C] Phe-tRNA^Phe or [³H] Tyr-tRNA^Phe. For poly-Tyr synthesis 0, 5, 50, or 500 nM PheRS was included as indicated. PheRS was also added to poly-Phe synthesis reactions at the same concentrations, but no change in poly-Phe synthesis was observed (data not shown). (B) Scheme of competition for Tyr-tRNA^Phe between PheRS and EF-Tu. As k₁[PheRS] > k₃ [EF-Tu] and k₂ > > k₋₁, the majority of free-standing Tyr-tRNA^Phe is bound by PheRS and hydrolyzed, whereas only a small fraction is utilized by the ribosome in protein synthesis. (C) Model for concerted editing pathways. (Top) cis-editing pathway. Upon synthesis at the active site (AS), a fraction of Tyr-tRNA^Phe directly translocates to the editing site for hydrolysis, which is not accessible to EF-Tu. (Bottom) trans-editing pathway. Tyr-tRNA^Phe dissociates from PheRS and is competed for by EF-Tu and PheRS. Tyr-tRNA^Phe bound to the editing site is rapidly hydrolyzed to yield a very low level of EF-Tu-bound Tyr-tRNA^Phe in vivo. Error bars correspond to the standard deviation from three independent experiments.