A RNA polymerase III promoter drives expression of a transcription unit composed of a non-coding leader (NCL) sequence (from the major mitochondrial 5S rRNA isoform encoded by the 5SrRNA gene), a translation initiation sequence and the mt:ATPase6 open reading frame (ORF). The ORF has been engineered for mitochondrial translation and a cytosolic stop codon (that encodes the Trp residue in mitochondria) has been introduced at codon 20. The presence of the NCL sequence targets the RNA to the mitochondrion. The presence of the early cytosolic stop codon is designed to prevent expression of the full-length mt:ATPase6 ORF in the cytosol, while allowing expression of the full-length ORF in the mitochondrion.