Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.).

Canonical DNA and RNA polymerases all synthesize nucleic acids in the 5′–3′ direction by virtue of the attack of a 3′-hydroxyl from the growing polynucleotide chain on the 5′-triphosphate of an incoming NTP.1,2 Enzymes of the tRNAHis guanylyltransferase (Thg1)/Thg1-like protein (TLP) superfamily are the only known exceptions to this rule.3,4 Thg1/TLP enzymes use the reverse chemistry, promoting the 3′-hydroxyl of an incoming NTP to attack a 5′-triphosphate (or similarly activated 5′-end) on the growing polynucleotide chain, thus achieving nucleotide addition in the opposite (3′–5′) direction compared to all other known polymerases.3,5 In eukaryotes, Thg1 enzymes use the 3′–5′ addition reaction to add a single G residue (G–1) to the 5′-end of tRNAHis,3,6,7 while TLPs utilize 3′–5′ polymerase chemistry to repair 5′-truncated tRNAs, which is a required reaction during mitochondrial 5′-tRNA editing and is likely involved in additional physiological processes.4,8−10 Despite the fact that Thg1 enzymes share no obvious sequence similarity with any other known enzyme family, the crystal structures of human Thg1 and bacterial TLP revealed that Thg1 family enzymes utilize a conserved active site that is strikingly similar to that of canonical 5′–3′ DNA/RNA polymerases to catalyze the 3′–5′ addition reaction.11,12 This observation raises new questions about the relationship between 5′–3′ and 3′–5′ polymerase enzymes and the possibility of more widespread use of the 3′–5′ polymerase active site in biology. Transient kinetic assays have been developed to characterize each of the three chemical steps that comprise the single-nucleotide (G–1) addition reaction, which is observed with 5′-monophosporylated eukaryotic (A73-containing) tRNAHis substrates. These chemical steps are (1) activation of the 5′-monophosphorylated tRNA by adenylylation, (2) nucleotidyl transfer to add the incoming NTP to the activated 5′-end of the tRNA, and (3) removal of the 5′-pyrophosphate from the added G–1 nucleotide (Figure S1 of the Supporting Information).13 Thg1 enzymes can also catalyze 3′–5′ addition via an ATP-independent mechanism that bypasses the first of these three chemical steps and utilizes a 5′-triphosphorylated tRNA as the activated substrate for nucleotidyl transfer (Figure S1 of the Supporting Information). This ATP-independent reaction was first observed in vitro with 5′-triphosphorylated transcripts3 and also occurs during the 3′–5′ polymerase reaction, once the first nucleotide has been added to the 5′-end of the tRNA5 (Figure ​(Figure1B).1B). The transient kinetic assays, in combination with alterations of Thg1 active site residues, revealed information about the molecular function of two nucleotides visualized in the Thg1 structure.11,13 Despite this information, many questions about the molecular mechanism of Thg1 catalysis remain unanswered, including the positions of the bound tRNA substrate and the incoming NTP that is added to the 5′-end, the basis for selection of a non-WC templated G–1 versus Watson–Crick (WC) base-pairing nucleotides during tRNA repair, and the roles of multiple conformational changes that are likely to occur during the reaction. Figure 1 Alternative biochemical reactions catalyzed by SceThg1. (A) SceThg1 catalyzes non-Watson–Crick addition of G–1 to wild-type (A73-containing) tRNAHis. After addition of the G–1 residue, SceThg1 removes pyrophosphate from the 5′-end ... One unanswered question is the molecular basis for distinct biochemical properties associated with eukaryotic Thg1-type enzymes compared with their largely bacterial and archaeal TLP counterparts. Eukaryotic Thg1 enzymes such as Saccharomyces cerevisiae Thg1 (SceThg1, formerly termed yThg1) catalyze two different types of 3′–5′ addition reactions with similar kinetic efficiency, depending on the tRNAHis substrate (Figure ​(Figure11).5 The first reaction is non-WC base-paired addition of G–1 opposite A73 observed with wild-type tRNAHis (Figure ​(Figure1A),1A), and the second is WC-templated 3′–5′ polymerization of multiple G-C or C-G base pairs observed with tRNAHis variants (Figure ​(Figure1B).1B). TLPs, on the other hand, are exclusively template-dependent 3′–5′ polymerases and do not efficiently add the non-WC paired G–1 nucleotide.9,10 Moreover, SceThg1 does not polymerize multiple A-U or U-A base pairs with tRNAHis, while TLPs catalyze polymerization of all four WC base-paired nucleotides.5,10 A key feature of the eukaryote-specific G–1 addition activity is the ability of the enzyme to terminate addition after only a single G–1 nucleotide is added to wild-type (A73-containing) tRNAHis, despite the presence of the downstream C74 and C75 residues in the tRNA, which are used as the template for addition of multiple G residues in the C73-containing tRNAHis variants. This property of SceThg1 is the focus of this study. According to the proposed mechanism of 3′–5′ addition, the presence of a 5′-triphosphate could be a controlling factor for the ability of Thg1 to add more than one nucleotide to the 5′-end of an RNA. When Thg1 adds multiple nucleotides, it is proposed to use the 5′-triphosphorylated end from the previous nucleotide addition as the activated end for each subsequent nucleotide addition (Figure ​(Figure1B);1B); however, SceThg1 also catalyzes removal of the 5′-pyrophosphate from the added G–1 nucleotide, and hydrolysis of this activated end could consequently prevent further nucleotide addition (Figure ​(Figure1A).1A). Thus, the pyrophosphate removal and nucleotide addition activities of SceThg1 could compete for available 5′-triphosphorylated ends generated by G–1 addition, impacting the balance between single- and multiple-nucleotide addition reactions. To test whether differences in the efficiency of pyrophosphate removal versus nucleotide addition steps catalyzed by SceThg1 explain the differences between single- and multiple-nucleotide addition reactions observed with different tRNAHis substrates, we developed transient kinetic assays to directly measure pyrophosphate removal and polymerization activities with a series of substrates that contain different combinations of N–1:N73 base pairs, in the presence of various NTPs. Here we demonstrate that the varied abilities of SceThg1 to remove the 5′-pyrophosphate from each tRNAHis substrate correlate well with the observed addition of multiple nucleotides to some tRNA substrates, but single nucleotides to others. Thus, the existence of the 5′-pyrophosphate removal activity catalyzed by SceThg1 is an important trait that is required for control of the 3′–5′ addition reaction with tRNAHis.