A modified uridine in the first position of the anticodon of a minor species of arginine tRNA, the argU gene product, from Escherichia coli

Abstract

The argU (dnaY) gene product, a minor tRNAArg, from Escherichia coii has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M. (1987) Nucleic Acids Res. 15, 3184]. In the present study, argU tRNA was purified from E. coli A 19 strain and nucleoside N* was characterized by the TLC and HPLC analyses. Nucleoside N* was found to be different from any naturally occurring modified nucleosides. From unfractionated E. coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H‐NMR experiments. By the analyses of one‐dimensional and two‐dimensional NMR spectra, nucleoside N* was suggested to be 5‐methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U. Thus, the occurrence of mnm5U in mature tRNA was found for the first time. Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.

The Escherichia coli argU10 (Ts) Phenotype Is Caused by a Reduction in the Cellular Level of the argU tRNA for the Rare Codons AGA and AGG

“…The argU tRNA recognizes the AGA and AGG codons (34,44). The TCT anticodon sequence in the argU gene was replaced by CCT, so that it could recognize only the AGG codon.…”

“…The argU tRNA has a UCU anticodon, where the uridine in the first position is modified to 5-methylaminomethyluridine (34), and thus it recognizes both the AGA and AGG codons (44). The AGG codon is also translated by another minor tRNA Arg species with a CCU anticodon, while this argU tRNA is the only tRNA Arg species that decodes AGA.…”

“…The wild-type and mutant argU tRNA species were overproduced in W3110 harboring pDM1 at 37°C and in GM10 harboring pKC1 at 30°C, respectively. The tRNAs were extracted from the late-log-phase cells and were fractionated by high-performance liquid chromatography on a hydroxyapatite column (HA-1000; Tosoh) (34). Finally, the argU tRNAs were purified from the column fractions by polyacrylamide gel electrophoresis (PAGE) on a denaturing 20% polyacrylamide gel.…”

“…For example, the arginine codons CGU and CGC, which are used frequently in the Escherichia coli genome, are recognized by the major tRNA Arg species. On the other hand, a minor tRNA Arg species (tRNA 4 Arg ), encoded by the argU gene (8), recognizes the rare arginine codons AGA and AGG (34,44). The rare codon AGG is also decoded by another minor tRNA Arg with a CCU anticodon (tRNA 5 Arg ).…”

Reassigning Sense Codon AGA to Encode Noncanonical Amino Acids in Escherichia coli

“…GM10 contains the argU10(Ts) mutation, a G1‐to‐A1 transition at the 5′‐end of argU tRNA, which encodes for the endogenous tRNA UCU . This mutation generates a mismatched base pair at the terminus of the acceptor stem of endogenous , which leads to its defective interactions with elongation factor Tu and arginyl–tRNA synthetase, further causing low activity in codon translation …”