Characterization of a Thg1-Like Protein in Dictyostelium discoideum

The tRNAHis guanylyltransferase (Thg1) is an essential, highly conserved enzyme in many eukaryotes, and the cytoplasmic role for members of this enzyme family has been well established and extensively studied; the Thg1 enzyme is well known to use an unusual 3'-5' polymerase activity to play an essential role in maturation of nucleus-encoded tRNAHis. However, recent studies in the slime mold Dictyostelium discoideum (Ddi-) have revealed new functions for Thg1 family members in mitochondrial metabolism that are not completely understood. This mold encodes two mitochondrial Thg1-Like Proteins (TLPs) that catalyze RNA modification by incorporating nucleotides in the same 3'-5' direction to distinct tRNA substrates, yet it does not encode the highly conserved RNase P in its mitochondria. I hypothesize that mitochondrial Thg1 homologs interact with other mitochondrial tRNA processing enzymes to catalyze their important functions, and possibly other functions that are distinct from the other more well-studied cytosolic functions of these enzymes. To test this hypothesis, I investigated the proteins that one of these homologs, DdiTLP2, interacts with during its catalysis of mitochondrial tRNAHis (mt-tRNAHis) maturation by using an epitope tag and immunoprecipitation. The epitope tagged protein was analyzed in vitro and in vivo, where an efficiency defect was discovered in both cases. Additionally, we have optimized a development assay for a deletion strain of DdiTLP2, discovering that it demonstrates a developmental defect.No embargoAcademic Major: Chemistr