Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System

The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34 ]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm5 ) or a 5-carbamoylmethyl (ncm5 ) side chain and sometimes an additional 2-thio (s2 ) or 2′-O -methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficientSaccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5 U34 - and some ncm5 U34 -containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.