In contrast to Dnmt2 methyltransferases from eukaryotes which methylate C38 of tRNAAsp, this enzyme methylates C38 in tRNAGlu. This swapped tRNA specificity is dependent on the differences in the sequence of the variable-loop in the substrate tRNAs (Shanmugam et al. 2011 ). Shanmugam and coauthors compared the secondary structures of tRNAAsp and tRNAGlu from several species
and noticed that Gs-tRNAAsp has a longer variable loop with extra guanine. Furthermore, the variable loops of all preferred Dnmt2 substrates (namely tRNAAsp
from all species but Geobacter and Gs-tRNAGlu) have at least one A at position 45 or 46 of the variable loop, while all the inactive counterparts contain a GG dinucleotide. Based
on this, the authors speculated that the variable loop may be a specificity determinant of GsDnmt2. To test this hypothesis, the variable loop of murine tRNAAsp was swapped with that of
murine tRNAGlu by an exchange of two nucleotides (AGAC to CGGC) which introduced the GG dinucleotide sequence into tRNAAsp. The mutant tRNAAsp was synthesized by in
vitro transcription. Equal amounts of the wild-type and mutant tRNAs were used in methylation reactions with GsDnmt2. Strikingly, the alteration
of these two bases in the variable loop of murine tRNAAsp led to a strong decrease in the methylation rate. This result supports the hypothesis of the involvement of the
variable loop in the enzyme-substrate recognition of GsDnmt2 (Shanmugam et al. 2011 ).