| N1 methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae , the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m 1G9 modification in yeast. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known. Likewise, nucleotide sequence shared between substrate tRNAs that might serve as a positive determinant for Trm10 recognition has not been identified. This dissertation focuses on the biochemical characterization of Trm10 substrate specificity and describes an expansion of substrates in vitro and under conditions of over-expression in vivo. Through testing a series of chimeric type II tRNAs, I show that a fully structured tRNA is required for recognition by Trm10, and that recognition specifically occurs in the D-stem/loop and variable loop of the tRNA. I posit potential roles for Trm10 promiscuity and discuss the loss of Trm10 function in the context of cellular stress and human disease. This body of work represents the first biochemical characterization of the tRNA metyltransferase Trm10 and highlights the importance of this non-essential, yet evolutionarily conserved, tRNA modification enzyme. |
