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The tRNAHis guanylyltransferase (Thg1) family of enzymes comprises members from all three domains of life (Eucarya, Bacteria, Archaea). Although the initial activity associated with Thg1 enzymes was a single 3′-to-5′ nucleotide addition reaction that specifies tRNAHis identity in eukaryotes, the discovery of a generalized base pair–dependent 3′-to-5′ polymerase reaction greatly expanded the scope of Thg1 family–catalyzed reactions to include tRNA repair and editing activities in bacteria, archaea, and organelles. While the identification of the 3′-to-5′ polymerase activity associated with Thg1 enzymes is relatively recent, the roots of this discovery and its likely physiological relevance were described ∼30 yr ago. Here we review recent advances toward understanding diverse Thg1 family enzyme functions and mechanisms. We also discuss possible evolutionary origins of Thg1 family–catalyzed 3′-to-5′ addition activities and their implications for the currently observed phylogenetic distribution of Thg1-related enzymes in biology.

Genes with sequence similarity to the yeast tRNAHis guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNAHis maturation to 5′-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3′-5′ nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G−1 addition enzyme likely to be responsible for tRNAHis maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5′-tRNA editing, based on their ability to efficiently repair the 5′ ends of mitochondrial tRNA editing substrates. Although 5′-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5′-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3′-5′ nucleotide addition reactions for diverse biological functions in the same organism.

The mitochondrial genome of Physarum polycephalum encodes five tRNAs, four of which are edited by nucleotide insertion. Two of these tRNAs, tRNAmet1 and tRNAmet2, contain predicted mismatches at the beginning (proximal end) of the acceptor stem. In addition, the putative 5′ end of tRNAmet2 overlaps the 3′ end of a small, abundant, noncoding RNA, which we term ppoRNA. These anomalies led us to hypothesize that these two Physarum mitochondrial tRNAs undergo additional editing events. Here, we show that tRNAmet1 and tRNAmet2 each has a nonencoded G at its 5′ end. In contrast to the other nucleotides that are added to Physarum mitochondrial RNAs, these extra G residues are likely added post-transcriptionally based on (1) the absence of added G in precursor transcripts containing inserted C and AA residues, (2) the presence of potential intermediates characteristic of 5′ replacement editing, and (3) preferential incorporation of GTP into tRNA molecules under conditions that do not support transcription. This is the first report of both post-transcriptional nucleotide insertions and the addition of single Gs in P. polycephalum mitochondrial transcripts. We postulate that tRNAmet1 and tRNAmet2 are acted upon by an activity similar to that present in the mitochondria of certain other amoebozoons and chytrid fungi, suggesting that enzymes that repair the 5′ end of tRNAs may be widespread.

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of non-encoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.