Interplay of heterogeneous transcriptional start sites and translational selection of AUGs dictate the production of mitochondrial and cytosolic/nuclear tRNA nucleotidyltransferase from the same gene in yeast

ATP (CTP):tRNA nucleotidyltransferase catalyzes the addition of the CCA end to tRNAs. In yeast, nucleotidyltransferase is encoded by the CCA1 gene and is localized to three cellular compartments: mitochondria, nucleus, and cytosol. There are three in-frame ATGs near the 5‘ end of the CCA1 open reading frame. Primer extension experiments show multiple transcription initiation sites upstream of ATG1 and between ATG1 and ATG2. Fractionation of cells carrying a CCA1-COXIV fusion gene demonstrates that all three in-frame AUGs are used as sites of initiation of translation. Therefore, both transcription of CCA1 mRNA with heterogeneous 5‘ ends and translation from downstream AUGs in CCA1 mRNAs play a role in the synthesis of three nucleotidyltransferase isozymes. Protein initiating from AUG1 is required for mitochondrial protein synthesis and, like many other proteins targeted to mitochondria, it is processed at the amino terminus upon import into the organelle. The shorter proteins arising from AUG2 and AUG3 provide nuclear/cytosol activity.