Functional analysis of tRNA modification enzymes using mutational profiling.

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA mA22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (mA), 3-methylcytosine (mC), 3-methyluridine (mU), 1-methylguanosine (mG), and N2,N2-dimethylguanosine (mG).