Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase - PubMed (original) (raw)
Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase
Hervé Roy et al. EMBO J. 2004.
Abstract
Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.
Figures
Figure 1
Tyr-dependent ATP hydrolysis by E. coli PheRS. Reactions were performed as described (15 μl samples) in the presence of 2 μM PheRS, with addition of tRNAPhe (2 μM) as indicated. Wild-type PheRS (▵), wild-type PheRS and tRNAPhe (▴), αA294G PheRS, (□), αA294G PheRS and tRNAPhe (▪). Inset, as main chart.
Figure 2
Specific deacylation of Tyr-tRNAPhe by E. coli PheRS. Reactions were performed as described (15 μl samples) in the presence of 2 nM PheRS and 1 μM Tyr-tRNAPhe. Wild-type PheRS (▴), αA294G PheRS (▪), no enzyme (◊). Inset, as main chart, except Phe-tRNAPhe (1 μM) was used instead of Tyr-tRNAPhe.
Figure 3
Mapping of conserved residues at putative editing sites in the β subunit of PheRS. (A) Schematic representation of the structure of T. thermophilus (αβ)2 PheRS complexed with tRNAPhe. (B) Representation of the coloring scheme used for mapping conservation onto the surface of the structure. (C) Surface map of eubacterial PheRS sequence conservation. (D) Enlarged section from C showing putative editing sites and their distances from A73 of tRNAPhe. The distance to the active site is also shown for comparison. Residue numbering for the corresponding positions in the E. coli enzyme is shown.
Figure 4
In vivo phenotypes of editing defective PheRS. (A) Schematic representation of the restoration of recoded β-galactosidase by editing defective PheRS. (B) Restoration of β-galactosidase activity in CC503 by PheRSαA294G/βA356W. (C, D) Editing-defective PheRS slows E. coli growth. E. coli containing plasmids encoding PheRSαA294G (•) or PheRSαA294G/βA356W (○) were grown until A595 nm was approximately 0.3, and then IPTG (1 mM) was added (indicated by ←) to increase the production of plasmid-encoded PheRS. Either complete minimal medium (C) or minimal medium lacking Phe and enriched in Tyr (D) were employed.
Figure 5
In vitro phenotypes of editing defective PheRS. (A) Hydrolysis of Tyr-tRNAPhe (1 μM) by PheRS (2 nM). (B) Aminoacylation of tRNAPhe (2.7 μM) with Tyr (30 μM) by PheRS (250 nM). Wild-type PheRS (▴), αA294G PheRS (▪), αA294G-βH265A (▵), αA294G-βE334A (•), αA294G-βA356W (▾), no enzyme (◊).
Figure 6
In vitro phenotypes of individual PheRS subunits. (A) Hydrolysis of Tyr-tRNAPhe (0.5 μM) by PheRS (12 nM). (B) Aminoacylation of tRNAPhe (2.4 μM) with Phe (20 μM) by PheRS (50 nM). Native αA294G/wild-type β subunit PheRS (▪), reconstituted αA294G/wild-type β subunit PheRS (▾), A294G α subunit (•), wild-type β subunit (▴), no enzyme (◊).
Figure 7
Model of the post-transfer editing site of PheRS. (A) Cross section of T. thermophilus PheRS in complex with tRNAPhe (1EIY). The CCA moiety of tRNAPhe (grey) is bound into the synthetic site of the α subunit of the enzyme. (B) Model for Tyr-tRNAPhe (red) binding in the editing site of the B3/B4 domain of the β subunit (see text, geometry of the model was optimized with DSviewer pro 5.0 (Accelrys)). tRNAPhe (grey) as found in the original structure is superimposed. (C) Cross section of the model of the B3/B4 domain bound to Tyr-tRNAPhe. The editing site is localized at the interface of domains B3 (in blue) and B4 (in yellow). (D) Ribbon representation of the model of the B3/B4 domain bound to Tyr-tRNAPhe. A76-Tyr is maintained between the two conserved motifs ‘GVMGGxxS/T' and ‘QPxHxFD'. Conserved residues in close contact with the Tyr moiety are displayed. Except for tRNAs and part (C), colors represent the percentage of identity for each position in an alignment of 103 eubacterial PheRSs (see Figure 3).
References
- Atlas RM (1993) Handbook of Microbiological Media. Boca Raton: CRC Press
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