Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli

Abstract

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA1 fMet, and a distantly located metY gene encodes a variant, tRNA2 fMet. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA2 fMet with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA + host background. The mutations were observed when the plasmid-borne gene encoded tRNA2 fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA1 fMet genes in E. coli in safeguarding their intactness, are discussed.

How Many Initiator tRNA Genes Does Escherichia coli Need?

“…To compare the fitness of strains carrying different numbers of tRNA i genes, we exploited E. coli Su15 (14). In this strain, metY is mutated to carry tRNA i with a CUA anticodon (instead of CAU), which disables initiation from the AUG or related codons in native mRNAs.…”

“…In this strain, metY is mutated to carry tRNA i with a CUA anticodon (instead of CAU), which disables initiation from the AUG or related codons in native mRNAs. However, the cell invests in making the tRNA and in its aminoacylation and formylation (14). This minimal change in metY avoids the polar effects sometimes associated with gene knockouts.…”

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

“…However, when the cells are supplied with the tRNA fMet whose anticodon is mutated to CUA (from CAU), initiation from the UAG start codon produces CAT protein conferring Cm R to the cell. 18,22,39 The tRNA fMet with CUA anticodon is aminoacylated with Gln by GlnRS. 18 When the 3GC pairs in the anticodon stem of the tRNA fMet are mutated to those found in the elongator tRNA Met (UA/CG/A, called 3GC mutant tRNA fMet ), the mutant tRNA loses its competence to initiate even though it is efficiently aminoacylated and formylated, rendering the host Cm sensitive (Cm S ).…”

An orthogonal amber initiator tRNA functions similarly across diverse <em>Escherichia coli</em> laboratory strains

“…coli encodes two sequence variants of the initiator tRNA, called tRNA fMet1 and tRNA fMet2 . The difference between these sequence variants is one nucleotide at position 46 where tRNA fMet1 is a G (modified to 7 mG in tRNA) and tRNA fMet2 is an A [17]. Both the tRNA fMet1 (metZWV genes) and tRNA fMet2 (metY gene) variants are present in the E. coli K-12 strains, but other strains of E. coli lack the tRNA fMet2 and instead have a fourth copy of tRNA fMet1 in the metY loci in their genomes (Fig.…”

“…(B) metZWV and metY loci in E. coli strains. Initiator tRNA fMet1 and tRNA fMet2 differ in sequence by a single nucleotide at position 46 in the variable loop, where tRNA fMet1 has 7 mG and tRNA fMet2 has an A [17]. The dashed lines represent similar genomic loci in each strain while boxes schematically represent the variation of tRNA fMet1 and tRNA fMet2 placement within each locus.…”