A glycine hinge for tRNA-dependent translocation of editing substrates to prevent errors by leucyl-tRNA synthetase

Abstract

Aminoacyl-tRNA synthetases often rely on a proofreading mechanism to clear mischarging errors before they can be incorporated into newly synthesized proteins. Leucyl-tRNA synthetase (LeuRS) houses a hydrolytic editing pocket in a domain that is distinct from its aminoacylation domain. Mischarged amino acids are transiently translocated approximately 30A between active sites for editing by an unknown tRNA-dependent mechanism. A glycine within a flexible beta-strand that links the aminoacylation and editing domains of LeuRS was determined to be important to tRNA translocation. The translocation-defective mutation also demonstrated that the editing site screens both correctly and incorrectly charged tRNAs prior to product release.

Fig. 1

Tertiary and primary structures of LeuRS. (a) Crystal structure of T. thermophilus LeuRS (PDB code 2BTE) [16]. The enzyme is displayed as a grey ribbon, with the CP1 domain inserted into the canonical core by two β-strands (black). (b, c) Sequence alignment of the N- and C-terminal β-strand linkers. Black and grey shading highlight highly conserved and homologous residues, respectively. The N- and C-terminal β-strands are marked by horizontal arrows and the glycine residues that were mutated are indicated by vertical arrows.

Fig. 2

Aminoacylation and deacylation activities. (a) Leucylation activities were obtained using 50 nM enzyme and 4 μM tRNA^Leu. (b) Deacylation reactions contained 100 nM enzyme and approximately 4 μM [³H]-Ile-tRNA^Leu.

Fig. 3

Misaminoacylation activity. Reactions contained 1 μM enzyme and 4 μM tRNA^Leu.

Fig. 4

Rescue of leucylation activity by T252A double mutant LeuRSs. (a) Aminoacylation reaction catalyzed by wild type LeuRS showing release of Leu-tRNA^Leu. (b) Aminoacylation reaction catalyzed by T252A LeuRS showing editing of correctly charged Leu-tRNA^Leu. [12] (c) Leucylation activities were obtained using 50 nM enzyme and 4 μM tRNA^Leu. (d) Deacylation activities were obtained using 100 nM enzyme and approximately 4 μM [³H]-Leu-tRNA^Leu.

Fig. 5

Proposed LeuRS reaction pathway and translocation determinant. The Gly229 residue is located within the N-terminal β-strand linker. The T252A mutation uncouples specificity in the editing active site [12].