Pre-transfer Editing by Class II Prolyl-tRNA Synthetase

Abstract

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3 end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of posttransfer editing, or the N-or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.

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

“…1B, Sc ⌬N183 ProRS).To establish whether the N terminus of yeast ProRS contributes to aminoacylation activity, the ⌬N183 variant was tested for its ability to charge a WT Sc tRNA Pro transcript in vitro. Interestingly, the k cat /K M for cognate proline charging was only 3-fold reduced relative to charging by WT Sc ProRS (9). Similar reductions were observed in the k cat /K M for proline (5-fold) and alanine (2-fold) activation upon deletion of the N-terminal domain (Table 1).…”

“…Here, we use mischarged Sc Ala-tRNA Pro to confirm that Sc ProRS appears to lack posttransfer editing function. We also show that the presence of the N-terminal domain plays a minor role in enhancing in vitro adenylate formation (Table 1) and tRNA aminoacylation (9). The specificity of amino acid activation (i.e., relative k cat /K M for cognate proline vs. alanine) is also enhanced Ϸ3-fold in vitro (Table 1).…”

“…WT Sc ProRS, ⌬N183 Sc ProRS, WT Deinococcus radiodurans ProRS, WT Ec AlaRS, and the Ec ProRS INS domain were purified using a Talon cobalt affinity resin (Clontech, Mountain View, CA) as described (9,43). Plasmids encoding the Ec/Sc chimera and the K279A Ec/Sc chimera were transformed into Ec SG13009 [pREP4] (Qiagen) competent cells and protein expression was induced with 0.1 mM isopropyl ␤-D-thiogalactoside for 22 h at room temperature.…”

“…In the case of some synthetases such as isoleucyl-tRNA synthetase, the pretransfer editing reaction has also been suggested to take place in the posttransfer editing domain (8). In contrast, in the case of prolyl-tRNA synthetase (ProRS), pretransfer editing appears to occur in the aminoacylation active site (9).…”

“…4). The extent of misacylation by the yeast enzyme is low, which indicates that pretransfer editing may be the predominant mode of clearing misactivated alanine by Sc ProRS (9). were replaced with residues 224-407 of the Ec INS domain (Fig.…”