Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase

Abstract

The accurate expression of genetic information relies on the fidelity of amino acid-tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs.

Figure 1

Schematic presentation of enzymatic reactions catalyzed by IleRS. The central pathway represents amino acid activation, tRNA binding, aminoacyl transfer, and dissociation of aminoacylated tRNA from the enzyme. The synthetic pathway may occur with both cognate and noncognate amino acids. Editing pathways are shown to the left and to the right. Pretransfer editing may proceed through enhanced dissociation of noncognate aminoacyl-AMP (1) or through its enzymatic hydrolysis, which may be tRNA-independent (2) or tRNA-dependent (3). Misacylated tRNA is deacylated through post-transfer editing (4).

Figure 2

Sequence alignment of a Rossmann fold peptide directly N-terminal to the strictly conserved HIGH motif, in various MetRS, LeuRS, and IleRS enzymes. In the case of MetRS and LeuRS, the alignment contains sequences from prokaryotes (E. coli, Staphylococcus aureus, and Bacillus subtilis), archaea (Pyrococcus horikoshii), and eukaryotes (Saccharomyces cerevisiae and Homo sapiens). The alignment additionally contains the sequence of the second IleRS from S. aureus and further sequences from prokaryotes Pseudomonas aeruginosa and Streptomyces griseus. The residue mutated in this work is marked with an asterisk.

Figure 3

Overlapped structures of E. coli LeuRS in complex with 5′-O-[N-(l-leucyl)sulfamoyl]adenosine (Leu-AMS) and tRNA^Leu (Protein Data Bank entry 4AQ7, colored green) and S. aureus IleRS in complex with mupirocin and tRNA^Ile (Protein Data Bank entry 1FFY, colored blue). Leu-AMS is colored yellow, the tRNA^Leu backbone orange, and its last nucleotide (A76) purple. tRNA^Ile and mupirocin are not visible. Tyrosine residues (Y43 of E. coli LeuRS and Y58 of S. aureus IleRS, both homologous to Y59 of E. coli IleRS) are shown as sticks. Superposition was done on the polypeptide backbone of parts of Rossmann folds (residues 34–94 and 619–666 in LeuRS and residues 44–104 and 600–647 in IleRS). The root-mean-square deviation was 0.885 Å.