Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli

Abstract

Mutations in the anticodon of tRNA Clu (UUC) were isolated or constructed and characterized for their ability to suppress cognate nonsense or missense mutations in vivo. The C36-to-A36 transversion mutation was isolated as an ochre and an amber suppressor, while the G36 transversion was selected as a CAG missense suppressor, tRNA G~u suppressors of an AAG missense mutation could not be isolated, and a 1536 transition mutation introduced into tRNA G~u in vitro conferred no suppressor phenotype. Over-expression of glutamyl-tRNA synthetase did not increase the activity of the U36 mutant tRNA clu, suggesting a defect at the level of translation rather than at the level of synthetase recognition.Key words: Suppressor tRNA; Translational efficiency; tRNAG~U; Anticodon a GAG(glu) to AAG(lys) missense mutation at the same position in lacZ were unsuccessful. A tRNA c~u allele containing a C-to-U transition at position 36 constructed by oligonucleotide-directed mutagenesis failed to suppress the same lacZ AAG missense mutation. The activity of the U36 mutant tRNA ~u, measured as fl-galactosidase activity, was not increased by over-expression of glutamyl-tRNA synthetase (GluRS). While defects in aminoacylation cannot be excluded, our observations are consistent with previous findings [6,7] and indicate that mutations at position 36 in the anticodon of tRNA Clu fail to produce efficient translational suppressors due primarily to a defect in codon recognition or tRNA-ribosome interaction. Materials and methods

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity

“…In contrast, Ec GluRS recognizes the G1:C72 and U2:A71 base pairs in the acceptor stem, A37 in the anticodon loop, U11:A24 base pair in D arm, and the U13:G22-A46 tertiary base pair in Ec tRNA Glu . Further, the lack of a nucleotide at position 47 and the modified uridine (s 2 U) in the UUC anticodon of tRNA Glu are also known identity determinants [33,34,37,38,39]. Ec tRNA Glu does contain several tRNA Asp identity elements: The G73 discriminator base, G10 in the D arm, and C38 in the anticodon loop.…”