A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

Abstract

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo 5U) † † Abbreviation used: U, uridine; m 5U, 5-methyluridine (ribothymidine); Um, 2′- O-methyluridine; mam 5s 2U, 5-methylaminomethyl-2-thiouridine; C, cytidine; Cm, 2′- O-methylcytidine; G, guanosine; m 1G, 1-methylguanosine; m 7G, 7-methylguanosine; A, adenosine; m 2A, 2-methyladenosine; m 6A, 6-methyladenosine; Am, 2′- O-methyladenosine; Gm, 2′- O-methylguanosine; cmo 5U, uridine-5-oxyacetic acid: mcmo 5U, methyl ester of cmo 5U; mo 5U, 5-methoxyuridine; mcm 5s 2U, 5-methoxycarbonylmethyl-2-thio-uridine; hU, dihydrouridine; ψ, pseudouridine. ho 5U, 5-hydroxyuridine. and its methylester, mcmo 5U. Both became radioactively labelled using [ methyl- 14C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo 5U and, in some cases, (4) mcmo 5U. The mutant had also become Aro −, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo 5U and mcmo 5U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo 5U and mcmo 5U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K 2 or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.