All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA His guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases. G-1 addition | reverse polymerase | tRNA modificationA ll nucleotide polymerases, including DNA and RNA polymerases, reverse transcriptase, and telomerase, catalyze nucleotide addition in the 5′ to 3′ direction. The reaction involves the nucleophilic attack of a polynucleotide terminal 3′-OH onto the α-phosphate of an incoming nucleotide, followed by release of the pyrophosphate moiety. Although the 5′ to 3′ direction has been adopted by all polymerases and transferases described to date, there is one notable exception: the enzyme tRNA His guanylyltransferase (Thg1). Thg1 catalyzes the highly unusual 3′-5′ addition of a single guanine to the 5′-end of tRNA His (1, 2). This reaction is an obligatory step in the maturation of this tRNA because the extra 5′ base, G −1 , constitutes a primary identity element for the aminoacyl-tRNA synthetase (HisRS) that attaches the amino acid histidine to the 3′-end of the tRNA (3-9). Thg1 is thus essential for maintaining the fidelity of protein synthesis. Consistent with the critical nature of the G −1 residue, THG1 is an essential gene in yeast and RNAi-mediated silencing of the Thg1 homolog in human cells results in severe cell-cycle progression and growth defects (2, 10, 11). Thg1 is widely conserved throughout eukarya, and Thg1 homologs are present in many archaea and bacteria.In eukarya, G −1 addition occurs opposite a universally conserved A 73 and thus is the result of a nontemplated 3′-5′ addition reaction. In addition, yeast Thg1 catalyzes a second reaction in vitro, extending tRNA substrates in the 3′-5′ direction in a template-directed manner driven by Watson-Crick pairing (12). Thg1 enzymes in archaea also catalyze template-dependent 3′-5′ addition, but do not catalyze nontemplated G −1 addition (13), suggesting that the templated 3′-5′ addition reaction likely represents an ancestral activity of the earliest Thg1 family members.The 3′-5′ addition of G −1 to tRNA His occurs via three chemical reactions, all catalyzed by Thg1 (2, 14) (Fig. 1). First, the 5′-monophosphorylated tRNA that results from RNase P cleavage of pre-tRNA His is activated using ATP, creating a 5...

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNAHis guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G−1 to the 5′-end of tRNAHis, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNAHis. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNAHis maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

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