The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Abstract

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo(5)UAC) contains a unique modification, N(6)-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA(1)(Val). Inactivation of yfiC gene abolishes m(6)A formation in tRNA(1)(Val), while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA(1)(Val) can be methylated by recombinant YfiC protein in vitro. Although the methylation of m(6)A in tRNA(1)(Val) by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.

FIGURE 1.

Methylation of tRNA species by recombinant YfiC protein in vitro. [³H]-SAM radioactivity introduced into various tRNA species by recombinant YfiC protein is indicated. (A) Methylation of tRNA species affinity purified from the cells. Gray bars correspond to methylation of tRNAs extracted from the wild-type strain, i.e., premethylated with nonradioactive SAM. Black bars correspond to methylation of tRNAs extracted from the yfiC knockout strain. K corresponds to mock tRNA purification with specific oligodeoxyribonucleotide omitted. (B) Methylation of in vitro transcribed tRNA species. Error bars indicate SD borders.

FIGURE 2.

MALDI MS analysis of the T1 digest of the tRNA₁^Val. Heavy part of the spectrum is shown. MALDI spectrum corresponding to fragments of the tRNA₁^Val from the (A) ΔyfiC strain, (B) wild-type strain, (C) ΔyfiC strain transformed with pCA24YfiC, and (D) ΔyfiC strain treated with SAM and recombinant YfiC in vitro. Oligonucleotides corresponding to the peak are indicated.

FIGURE 3.

Doubling time of the wild-type (gray bars) and ΔyfiC (black bars) strains. Growth conditions, such as temperature and medium, are indicated below the bars. Error bars indicate SD borders.

FIGURE 4.

Cell titer drop (cfu indicates colony forming units) in the logarithmic scale after exposure of the culture to various stress conditions. Stress type is indicated above the bars. Gray bars correspond to the wild-type strain; black bars correspond to the ΔyfiC strain. Error bars indicate SD borders.

FIGURE 5.

Highly expressed gene codon bias for tRNA₁^Val GUA (A) and GUG (B) codons. Plotted is the particular codon frequency (in percent relative to total codons) against total number of valine codons (in percent relative to total codons). Gray boxes correspond to each of E. coli genes. Black triangles correspond to highly expressed genes (Karlin et al. 1998, 2001). (C) Two-dimensional protein gel, showing a comparison of protein composition of wild-type strain (labeled red) and ΔyfiC strain (labeled green). Yellow color corresponds to equal amount of protein in both strains.