Co-evolution of bizarre armless tRNAs and their maturation enzymes in Enoplea

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In mitochondria of metazoans, many tRNAs show deviations from the standard structure, resulting in unusual and bizarre transcripts. In Enoplea mitochondria, we could recently identify dramatically reduced armless tRNAs, representing the most extreme form of this RNA type. To recognize these transcripts as substrates, mitochondrial tRNA maturation and aminoacylation activities must have undergone a specific adaptation. Using Romanomermis culicivorax as a model organism, we will characterize the corresponding mitochondrial CCA-adding enzyme, RNase P as well as several aminoacyl-tRNA synthetases as recombinant proteins. While the latter activities represent specialized mitochondrial enzyme versions, a single form of the CCA-adding enzyme is responsible for the maturation of both cytosolic (canonical) and mitochondrial (bizarre) tRNAs. Hence, this enzyme had to undergo an unusual co-evolution with its substrates. Using kinetic characterization, competition studies and the analysis of enzyme chimeras, we want identify this adaptation in the functional domains of the enzyme. In addition, individual R. culicivorax enzymes for tRNA precursor transcript processing and aminoacylation will be compared in their substrate specificity to activities acting on conventional tRNAs. Finally, structure analyses like SHAPE, in line probing as well as NMR studies of armless tRNAs will identify specific features required for the interplay between these bizarre transcripts and their maturation enzymes. These analyses will give detailed insights into the flexibility and adaptability of tRNA processing enzymes concerning the interaction with their substrates.