Identification and characterization of archaeal and fungal tRNA methyltransferases

All organisms modify their tRNAs by use of evolutionarily conserved enzymes. Members of the Archaea contain an extensive set of modified nucleotides that were early evidence of the fundamental evolutionary divergence of the Archaea from Bacteria and Eucarya. However, the enzymes responsible for these posttranscriptional modifications were largely unknown before the advent of genome sequencing. This chapter explains methods to identify tRNA methyltransferases in genome sequences, emphasizing the identification and characterization of six enzymes from the hyperthermophilic archaeon Methanocaldococcus jannaschii. We describe methods to express these proteins, purify or synthesize tRNA substrates, measure methyltransferase activity, and map tRNA modifications. Comparison of the archaeal methyltransferases with their yeast homologs suggests that the common ancestor of the archaeal and eucaryal organismal lineages already had extensive tRNA modifications.