Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.

Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa Are Individually Implicated in the Recognition of tRNA Asn

“…Then, the genes coding for the three subunits of the AdT could simply be removed from the chromosome, if not needed, provided that the pathway of synthesis of Gln-tRNA Gln is also a direct one, involving a GlnRS. As reported before, a small decrease (11,18) or increase (21) in the efficiency of an aaRS can lead to a detectable phenotype. In the case of H31L and G83K variants, 3.5-fold and 4.2-fold increases in the specificity of the enzyme, respectively, were enough to abolish the toxicity of the enzymes in vivo.…”

Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences

“…Despite strong conservation and ubiquitous presence of the Dnmt2 tRNA methyltransferase, the molecular function of the C38 methylation of tRNA Asp introduced by Dnmt2 remains unknown. Searching for a direct molecular function of this tRNA methylation, we have focused on its potential role in modulating the interaction with the aspartyl-tRNA synthetase (AspRS), because modifications in the anticodon loop of tRNAs have been shown to affect their charging in other cases [ 24–26 ], and C38 has been mapped as a direct identity determinant of AspRS from species in all kingdoms of life [ 23 ]. To investigate the role of C38 methylation of tRNA Asp in tRNA charging, we have cloned mouse AspRS from complementary DNA and purified the recombinant enzyme from E. coli [ 27 , 28 ] ( Supplementary Figure S1a ).…”

tRNAHis guanylyltransferase adds G−1 to the 5′ end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases

“…Synthetases often recognize the anticodon by direct hydrogen bonding between amino acid residues in the enzyme and nucleotides of the anticodon (Cusack et al 1998;Sekine et al 2001). These interactions can be powerful enough for a single amino acid to determine specificity for a given tRNA species (Sekine et al 2001;Feng et al 2003Feng et al , 2005, although, in some cases, other regions of the tRNA can play an important role (Cusack et al 1998;Martin et al 2004). The mechanism by which Thg1 recognizes the anticodon of its tRNA His substrate remains unclear, because Thg1 contains no identifiable similarity to tRNA synthetases, including in particular the known tRNA recognition motifs, and because important substrate recognition motifs of Thg1 have not yet been identified.…”