Isolation and Characterization of MODS, a Gene Required for Isopentenylation of Cytoplasmic and Mitochondrial tRNAs of Saccharomyces cerevisiae

Abstract

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N⁶-(Δ²-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to < 1.5% of wild-type levels. The tRNA modification enzyme, Δ²-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores Δ²-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N⁶-(Δ²-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N⁶-(Δ²-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for Δ²-isopentenyl pyrophosphate:tRNA isopentenyl transferase.