The YqfN protein of Bacillus subtilis is the tRNA: m 1 A22 methyltransferase (TrmK)

Abstract

N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.

m1A Post‐Transcriptional Modification in tRNAs

“…The m 1 A modification in nucleotide position 14 (m 1 A14) is rare, and has so far only been identified in (cyt)tRNA Phe from mammals [17,18], whereas the m 1 A22 modification has been identified only in tRNAs from bacteria [19,20,21,22]. The m 1 A57 modification was identified in archaea and exists only transiently as an intermediate leading to 1-methylinosine (m 1 I) by hydrolytic deamination [23,24].…”

Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)

“…Thus, eukaryotic Trm6-Trm61, eubacterial TrmI, and aTrmI have different protein architectures even though these enzymes are responsible for the same m 1 A58 modification in tRNA. In addition, recently, the enzymes responsible for m 1 A22 in eubacterial tRNA, m 1 A9 in human mitochondrial tRNA, m 1 G9 and m 1 A9 in archaeal tRNA, and m 1 A58 in mitochondrial tRNA were identified as TrmK (28), a subcomplex of mitochondrial RNase P (29), an archaeal Trm10 homolog (30), and Trm61B (31), respectively.…”

Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis

“…This modification brings a positive charge on the adenine aromatic ring. We focused our work on the methyltransferase TrmK from B. subtilis that catalyzes the methylation of adenine 22 of tRNA Ser and tRNA Tyr (Roovers et al 2008 …”

“…Recombinant B. subtilis TrmK was expressed and purified with a protocol adapted from Roovers et al (Roovers et al 2008). Since wild-type TrmK is prone to aggregation through cysteine oxidation, we produced a protein variant with cysteine to serine mutations (i.e.…”