Characterization of Thg1-like Proteins in tRNA Biogenesis

Abstract

Transfer RNAs (tRNAs) function to translate the nucleic acid code of messenger RNA into amino acid sequences, and the highly conserved secondary and tertiary structures of tRNAs are critical for proper recognition by the translational machinery. In all domains of life, the evolutionary pressure to maintain tRNA structure has led to diverse pathways in tRNA biogenesis that make use of a 3'-to-5' RNA polymerization reaction catalyzed by the tRNAHis guanylyltransferase (Thg1) enzyme family. Here, we study two processes catalyzed by Thg1 enzymes. First, members of the Thg1 enzyme family, designated as Thg1-like proteins (TLPs), have been suggested to participate in a multistep tRNA editing pathway in several lower eukarya. Organisms such as Dictyostelium discoideum and Acanthamoeba castellanii encode in their mitochondria aberrant tRNAs with mismatches at the normally base-paired tRNA acceptor stem. Two TLPs in D. discoideum (DdiTLP3-4) have robust in vitro 3'-to-5' polymerization on seven truncated tRNA intermediates proposed to be edited in D. discoideum, and the sequence of one tRNA product reaffirms the previously observed preference for templated nucleotide addition. Although DdiTLP3 and DdiTLP4 share similar tRNA repair activities, their mechanisms for repairing truncated tRNAs differ. DdiTLP3 is more sensitive to tRNA length and can only incorporate nucleotides to the first three positions of the 5' end of tRNAs. In contrast, DdiTLP4 repairs larger 5'-end truncations than DdiTLP3, yet it also catalyzes the incorporation of excess nucleotides at the 5' end under sub- physiological ATP concentrations. The second process we studied is the catalysis of G-1 addition to tRNAHis, a process thought to occur in all Eukarya and many Archaea. In A. castellanii, however, the two encoded TLPs (AcaTLP1/2) have very weak in vitro G-1 addition activity, and are likely involved in tRNA editing only. In studying D. discoideum and A. castellanii Thg1 enzymes, the activities and mechanistic properties of 3'-5' RNA polymerization provide insight in the evolution of the Thg1 superfamily and the roles they have adopted for tRNA biogenesis.