Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

Abstract

Aminoacyl-tRNA synthetases are multidomain proteins responsible for the attachment of specific amino acids to their tRNA substrates. Prolyl-tRNA synthetases (ProRSs) are notable due to their particularly diverse architectures through evolution. For example, Saccharomyces cerevisiae ProRS possesses an N-terminal extension with weak homology to a bacterial-specific domain typically present as an insertion (INS) within the aminoacylation active site. The INS domain has been shown to contain a "posttransfer" editing active site responsible for cleaving the aminoacyl-ester bond of misacylated Ala-tRNA(Pro) species. However, wild-type S. cerevisiae ProRS does not perform posttransfer editing in vitro. Here, we show that replacement of the N-terminal domain of S. cerevisiae ProRS with the Escherichia coli INS domain confers posttransfer editing function to this chimeric enzyme, with specificity for yeast Ala-tRNA(Pro). In contrast, the isolated INS domain displays only weak editing activity and lacks tRNA sequence specificity. These results emphasize the modular nature of synthetase editing active sites and demonstrate how in evolution, a weak editing activity can be converted to a more robust state through fusion to the body of a synthetase. In this manner, a single editing module can be distributed to different synthetases, and simultaneously acquire specificity and enhanced activity.

Fig. 1.

Schematic representation of the domain architecture of ProRS enzymes. (A) ProRS enzymes from all three domains of life. The class II consensus motifs and anticodon domains are shown in black and the bacterial INS domain is indicated as a white box. The C-terminal extension domains present in eukaryotic and archaeal ProRSs, which are not homologous to the INS domain, are shown in gray. The N-terminal extension unique to lower eukaryotic ProRSs is indicated as a white box with black stripes to indicate weak homology with the bacterial INS domain. (B) ProRS constructs used in this work: Sc ProRS with a deletion of the N-terminal 183 residues (Sc ΔN183 ProRS, Top); chimera with the Ec INS domain substituted for the first 183 aa of Sc ProRS (Ec/Sc ProRS, Middle); the Ec ProRS INS domain (Ec Insertion, Bottom).

Fig. 2.

Homology models. Models of the Ec INS domain (Right) and the Sc ProRS N terminus (Left) generated by using the known x-ray structure of the Hi YbaK protein (27).

Fig. 3.

Deacylation by WT Sc ProRS. Deacylation of Sc U70/A73-[³H]Ala-tRNA^Pro (filled circle) or Sc [³H]Pro-tRNA^Pro (filled square) by 4 μM Sc ProRS. A background (no protein) reaction was performed for all tRNAs tested and all were within 11% of the representative background data shown (open circle). An additional reaction (filled inverted triangle) was performed by incubating Sc U70/A73-[³H]Ala-tRNA^Pro with 0.2 M NaOH (pH 13). The plots represent the average of at least three assays.

Fig. 4.

Aminoacylation assays. Reaction time courses showing charging of alanine onto Sc tRNA^Pro by WT Sc ProRS (open circle), Ec/Sc chimera (open triangle), and K279A-Ec/Sc chimera (filled triangle). Assays were performed at 30°C using 12 μM enzyme and 8 μM WT Sc tRNA^Pro. The plots represent the average of two assays with the values differing by <15%.

Fig. 5.

Deacylation by the Ec/Sc chimera and the Ec INS domain. (A) Deacylation of Sc U70/A73-[³H]Ala-tRNA^Pro (filled inverted triangle), Sc [³H]Pro-tRNA^Pro (filled square), and Ec [³H]Ala-tRNA^Pro (open triangle) in the presence of Ec/Sc chimeric ProRS (1 μM). (B) Deacylation of Sc U70/A73-[³H]Ala-tRNA^Pro (open triangle), Sc [³H]Pro-tRNA^Pro (filled square), and Ec [³H]Ala-tRNA^Pro (filled triangle) in the presence of the Ec INS domain (20 μM). A background (no protein, open circle) reaction was performed for all tRNAs tested, and all of the plots present the average of at least three assays.