The balance between pre- and post-transfer editing in tRNA synthetases

Abstract

The fidelity of tRNA aminoacylation is dependent in part on amino acid editing mechanisms. A hydrolytic activity that clears mischarged tRNAs typically resides in an active site on the tRNA synthetase that is distinct from its synthetic aminoacylation active site. A second pre-transfer editing pathway that hydrolyzes the tRNA synthetase aminoacyl adenylate intermediate can also be activated. Pre- and post-transfer editing activities can co-exist within a single tRNA synthetase resulting in a redundancy of fidelity mechanisms. However, in most cases one pathway appears to dominate, but when compromised, the secondary pathway can be activated to suppress tRNA synthetase infidelities.

Fig. 1

Futile charging cycle. Uncoupling amino acid specificity mechanisms between the synthetic and hydrolytic active sites of an editing aaRS results in ATP depletion. In this example, substitution of a conserved threonine residue with alanine in the amino acid binding pocket of the editing active site allows binding of Leu-tRNA^Leu, which is then rapidly deacylated [10]. The conserved threonine sterically clashes with the γ-methyl branch of the leucine side chain (which is unique amongst the standard amino acids) to block binding by correctly charged Leu-tRNA^Leu [10,18].

Fig. 2

Aminoacylation and amino acid fidelity pathways: The aaRSs activate amino acid (aa) by forming an aminoacyl adenylate intermediate and then the amino acid is transferred to the cognate tRNA^aa isoacceptor. When a non-cognate amino acid (xx) is misactivated, it might be cleared by a pre-transfer editing mechanism that hydrolyzes the transient aminoacyl adenylate intermediate. Alternatively, the mischarged xx-tRNA^aa is deacylated by the aaRS in a post-transfer editing mechanism.

Fig. 3

Shift between redundant pre- and post-transfer editing pathways. A line of an arbitrary, non-linear scale is used to schematically represent the partition between pre- and post-transfer editing activities of a single aaRS. Arrows to the line indicate the predominance of one over the other alternative editing pathways. (A) Partition of wild-type aaRS pre- and post-transfer editing activities. (B) Shift of E. coli LeuRS editing mechanisms by introduction of a T252Y mutation to block post-transfer editing activity [51] and substitution of Ala 293 with aspartic acid (A293D) to activate pre-transfer editing [20]. (C) Shift of the editing partition for E. coli LeuRS using the double mutation T252Y/K186E [20]. (D) Complete shift of the editing partition from post-transfer to pre-transfer editing by deletion of the LeuRS CP1 domain (ΔCP1), which contains the tRNA deacylation active site [22].