Investigation of the Specificity of Modification Enzymes

We previously examined the specificity for G_-1 addition to tRNA^His and the specificity of tRNA variants for RTD, and recently we have focused on the specificity of modification enzymes for C₃₂.  We are interested in the C₃₂ modification because this residue is in the anticodon loop and is implicated in translation, and because C₃₂ can have three different fates in different tRNAs: it is modified to m³C in 6 tRNA species, it is modified to Cm in 3 tRNAs, and it is unmodified in 14 other tRNA species.

Our recent results show that the specificity of S. cerevisiae Trm140 for m³C modification is complex.  Trm140 recognizes and modifies its three tRNA^Thr substrates (with anticodons IGU, CGU, and UGU) based entirely on recognition of the anticodon residues G₃₅ and U₃₆ and the adjacent t⁶A₃₇ residue  Mutation of any of any of these residues eliminates modification in vivo and binding in vitro, and introduction of these residues to another tRNA is sufficient for both binding and modification.  Remarkably, however, Trm140 recognition of the three tRNA^Ser substrates (with anticodons UGA, CGA, and GCU) is completely different.  In this case, m³C modification requires seryl-tRNA synthetase and the distinctive variable loop of tRNA^Ser in vitro and in vivo, and is stimulated by t⁶A₃₇ or i⁶A₃₇ as well as by C₃₄ and G₃₅.  These seemingly very different modes of Trm140 recognition of tRNA^Thr and tRNA^Ser substrates appear to be widely conserved in Ascomycota, but Trm140 has duplicated into two or more paralogs in other fungi and in metazoans for separate modification of the two tRNA families.

Current work is focused on understanding the tRNA determinants that are recognized by Trm7 and its partner proteins, which also recognize C₃₂ of its substrate tRNAs, as well as the nearby anticodon residue N₃₄, and also have no obvious recognition elements in common.

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