YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA^Leu isoacceptors

Abstract

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 29-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA Leu CmAA and tRNA Leu cmnm5UmAA . Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 29-O-methylation at position 34 in both tRNA Leu CmAA and tRNA Leu cmnm5UmAA . Loss of YibK methylation reduces the efficiency of codon-wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized a/b knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 29-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N 6 -(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.

Bacillus subtilis exhibits MnmC-like tRNA modification activities

“…Bulk tRNA was purified as described [11]. Specific true native tRNAs and tRNAs overexpressed from pBSKrna-derived plasmids were purified from bulk tRNA by the Chaplet Column Chromatography method using biotinylated DNA probes immobilized on a HiTrap Streptavidin HP column [9,25].…”

“…The probes were complementary to the specific sequence of each tRNA (Supplementary Table S2). Analysis of nucleosides by reverse-phase HPLC was performed as described [11]. The nucleosides were identified according to their UV spectra, relative retention times, and by comparison with appropriate controls, including synthetic markers [7,9,26].…”

“…Notably, some tRNA substrates of MnmEG are also substrates of MnmA and TrmL (Figure 1). MnmA introduces the 2-thiol group into U34 of tRNA Glu UUC , tRNA Lys UUU and tRNA Gln UUG [10], whereas TrmL methylates the 2´-OH group of the U-ribose in tRNA Leu UAA [11]. Consequently, the final modifications in U34 are mnm 5 s 2 U in tRNA Lys UUU and tRNA Glu UUC , cmnm 5 s 2 U and, to a much lesser extent, mnm 5 s 2 U in tRNA Gln UUG , and cmnm 5 Um in tRNA Leu UAA [9].…”

Characterization of two homologous 2′-O-methyltransferases showing different specificities for their tRNA substrates

“…Paralogs of TrmH are found in various bacteria (Anantharaman et al 2002). These include TrmJ and TrmL, which catalyze the methylation of the ribose of nucleosides on, respectively, positions 32 and 34 (Purta et al 2006;Benitez-Paez et al 2010). Surprisingly, in Eukarya, methylation of the ribose moiety of nucleosides 32 and 34 is carried out by the completely unrelated Trm7 enzyme, assisted by either the auxiliary protein Trm732 or Trm734 (Pintard et al 2002;Guy et al 2012).…”

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

“…Also, by focusing on the m 1 G37 reaction, kinetic analysis can identify AdoMet analogs that specifically inhibit the reaction, rather than those that inhibit other AdoMet-dependent reactions. For example, analogs that target TrmD can be separated by cross-reactivity analysis from those that target TrmH (Nureki et al 2004) and TrmL (Benítez-Páez et al 2010), which adopt the same trefoil-knot structure as in TrmD but synthesize Gm18 and Um34 on tRNA, respectively.…”