Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.

Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNATrp, based on the inability of mutants of each enzyme to rescue the growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

Diversity in Biological Function and Mechanism of the tRNA Methyltransferase Trm10.

ConspectusTransfer ribonucleic acid (tRNA) is the most highly modified RNA species in the cell, and loss of tRNA modifications can lead to growth defects in yeast as well as metabolic, neurological, and mitochondrial disorders in humans. Significant progress has been made toward identifying the enzymes that are responsible for installing diverse modifications in tRNA, revealing a landscape of fascinating biological and mechanistic diversity that remains to be fully explored. Most early discoveries of tRNA modification enzymes were in model systems, where many enzymes were not strictly required for viability, an observation somewhat at odds with the extreme conservation of many of the same enzymes throughout multiple domains of life. Moreover, many tRNA modification enzymes act on more than one type of tRNA substrate, which is not necessarily surprising given the similar overall secondary and tertiary structures of tRNA, yet biochemical characterization has revealed interesting patterns of substrate specificity that can be challenging to rationalize on a molecular level. Questions about how many enzymes efficiently select a precise set of target tRNAs from among a structurally similar pool of molecules persist.The tRNA methyltransferase Trm10 provides an exciting paradigm to study the biological and mechanistic questions surrounding tRNA modifications. Even though the enzyme was originally characterized in Saccharomyces cerevisiae where its deletion causes no detectable phenotype under standard lab conditions, several more recently identified phenotypes provide insight into the requirement for this modification in the overall quality control of the tRNA pool. Studies of Trm10 in yeast also revealed another characteristic feature that has turned out to be a conserved feature of enzymes throughout the Trm10 family tree. We were initially surprised to see that purified S. cerevisiae Trm10 was capable of modifying tRNA substrates that were not detectably modified by the enzyme in vivo in yeast. This pattern has continued to emerge as we and others have studied Trm10 orthologs from Archaea and Eukarya, with enzymes exhibiting in vitro substrate specificities that can differ significantly from in vivo patterns of modification. While this feature complicates efforts to predict substrate specificities of Trm10 enzymes in the absence of appropriate genetic systems, it also provides an exciting opportunity for studying how enzyme activities can be regulated to achieve dynamic patterns of biological tRNA modification, which have been shown to be increasingly important for stress responses and human disease. Finally, the intriguing diversity in target nucleotide modification that has been revealed among Trm10 orthologs is distinctive among known tRNA modifying enzymes and necessitates unusual and likely novel catalytic strategies for methylation that are being revealed by biochemical and structural studies directed toward various family members. These efforts will no doubt yield more surprising discoveries in terms of tRNA modification enzymology.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/ß fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

Transient kinetic analysis for studying ionizations in RNA modification enzyme mechanisms.

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.

Fidelity of base-pair recognition by a 3'-5' polymerase: mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase.

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3'-5' addition of a single nontemplated guanosine (G-1) to the 5' end of tRNAHis In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3'-5' polymerization of Watson-Crick (WC) base pairs, utilizing nucleotides in the 3'-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3'-5' polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3'-5' polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA "template," addition of a non-WC G-1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5'-3' polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3'-5' polymerases.

PRMT5 regulates T cell interferon response and is a target for acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a T cell-mediated immunological disorder and the leading cause of nonrelapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that protein arginine methyltransferase 5 (PRMT5) and arginine methylation are upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity were upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression was also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared with those who did not develop aGVHD. PRMT5 inhibition using a selective small-molecule inhibitor (C220) substantially reduced mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors substantially improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retained the beneficial graft-versus-leukemia effect by maintaining cytotoxic CD8+ T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduced STAT1 phosphorylation as well as transcription of proinflammatory genes, including interferon-stimulated genes and IL-17. Additionally, PRMT5 inhibition deregulates the cell cycle in activated T cells and disrupts signaling by affecting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.

Distinct substrate specificities of the human tRNA methyltransferases TRMT10A and TRMT10B.

The tRNA m1R9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea and most single-celled eukaryotes, metazoans encode up to three homologs of Trm10. Several disease states correlate with a deficiency in the human homolog TRMT10A, despite the presence of another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena and demonstrate that human TRMT10A (hTRMT10A) and human TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays with purified hTRMT10A and hTRMT10B reveal a robust activity for hTRMT10B as a tRNAAsp-specific m1A9 methyltransferase and suggest that it is the relevant enzyme responsible for this newly discovered m1A9 modification in humans. Moreover, a comparison of the two cytosolic enzymes with multiple tRNA substrates exposes the enzymes' distinct substrate specificities, and suggests that hTRMT10B exhibits a restricted selectivity hitherto unseen in the Trm10 enzyme family. Single-turnover kinetics and tRNA binding assays highlight further differences between the two enzymes and eliminate overall tRNA affinity as a primary determinant of substrate specificity for either enzyme. These results increase our understanding of the important biology of human tRNA modification systems, which can aid in understanding the molecular basis for diseases in which their aberrant function is increasingly implicated.

Chemical footprinting and kinetic assays reveal dual functions for highly conserved eukaryotic tRNAHis guanylyltransferase residues.

tRNAHis guanylyltransferase (Thg1) adds a single guanine to the -1 position of tRNAHis as part of its maturation. This seemingly modest addition of one nucleotide to tRNAHis ensures translational fidelity by providing a critical identity element for the histidyl aminoacyl tRNA synthetase (HisRS). Like HisRS, Thg1 utilizes the GUG anticodon for selective tRNAHis recognition, and Thg1-tRNA complex structures have revealed conserved residues that interact with anticodon nucleotides. Separately, kinetic analysis of alanine variants has demonstrated that many of these same residues are required for catalytic activity. A model in which loss of activity with the variants was attributed directly to loss of the critical anticodon interaction has been proposed to explain the combined biochemical and structural results. Here we used RNA chemical footprinting and binding assays to test this model and further probe the molecular basis for the requirement for two critical tRNA-interacting residues, His-152 and Lys-187, in the context of human Thg1 (hThg1). Surprisingly, we found that His-152 and Lys-187 alanine-substituted variants maintain a similar overall interaction with the anticodon region, arguing against the sufficiency of this interaction for driving catalysis. Instead, conservative mutagenesis revealed a new direct function for these residues in recognition of a non-Watson-Crick G-1:A73 bp, which had not been described previously. These results have important implications for the evolution of eukaryotic Thg1 from a family of ancestral promiscuous RNA repair enzymes to the highly selective enzymes needed for their essential function in tRNAHis maturation.

A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.

5'-End sequencing in Saccharomyces cerevisiae offers new insights into 5'-ends of tRNAHis and snoRNAs.

tRNAHis guanylyltransferase (Thg1) specifies eukaryotic tRNAHis identity by catalysing a 3'-5' non-Watson-Crick (WC) addition of guanosine to the 5'-end of tRNAHis . Thg1 family enzymes in Archaea and Bacteria, called Thg1-like proteins (TLPs), catalyse a similar but distinct 3'-5' addition in an exclusively WC-dependent manner. Here, a genetic system in Saccharomyces cerevisiae was employed to further assess the biochemical differences between Thg1 and TLPs. Utilizing a novel 5'-end sequencing pipeline, we find that a Bacillus thuringiensis TLP sustains the growth of a thg1Δ strain by maintaining a WC-dependent addition of U-1 across from A73 . Additionally, we observe 5'-end heterogeneity in S. cerevisiae small nucleolar RNAs (snoRNAs), an observation that may inform methods of annotation and mechanisms of snoRNA processing.

The Role of 3' to 5' Reverse RNA Polymerization in tRNA Fidelity and Repair.

The tRNAHis guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3' to 5' synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3' to 5' chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed.

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis.

The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using S-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m¹G9 or m¹A9-specific enzymes, while others are bifunctional enzymes catalyzing both m¹G9 and m¹A9. This variability is particularly intriguing given different chemical properties of the target N1 atom of guanine and adenine. Here we performed an extensive kinetic and mutational analysis of the m¹G9 and m¹A9-catalyzing Trm10 from Thermococcus kodakarensis to gain insight into the active site that facilitates this unique bifunctionality. These results suggest that the rate-determining step for catalysis likely involves a conformational change to correctly position the substrate tRNA in the active site. In this model, kinetic preferences for certain tRNA can be explained by variations in the overall stability of the folded substrate tRNA, consistent with tRNA-specific differences in metal ion dependence. Together, these results provide new insight into the substrate recognition, active site and catalytic mechanism of m¹G/m¹A catalyzing bifunctional enzymes.

A Family Divided: Distinct Structural and Mechanistic Features of the SpoU-TrmD (SPOUT) Methyltransferase Superfamily.

The SPOUT family of enzymes makes up the second largest of seven structurally distinct groups of methyltransferases and is named after two evolutionarily related RNA methyltransferases, SpoU and TrmD. A deep trefoil knotted domain in the tertiary structures of member enzymes defines the SPOUT family. For many years, formation of a homodimeric quaternary structure was thought to be a strict requirement for all SPOUT enzymes, critical for substrate binding and formation of the active site. However, recent structural characterization of two SPOUT members, Trm10 and Sfm1, revealed that they function as monomers without the requirement of this critical dimerization. This unusual monomeric form implies that these enzymes must exhibit a nontraditional substrate binding mode and active site architecture and may represent a new division in the SPOUT family with distinct properties removed from the dimeric enzymes. Here we discuss the mechanistic features of SPOUT enzymes with an emphasis on the monomeric members and implications of this "novel" monomeric structure on cofactor and substrate binding.

Mechanistic features of the atypical tRNA m1G9 SPOUT methyltransferase, Trm10.

The tRNA m1G9 methyltransferase (Trm10) is a member of the SpoU-TrmD (SPOUT) superfamily of methyltransferases, and Trm10 homologs are widely conserved throughout Eukarya and Archaea. Despite possessing the trefoil knot characteristic of SPOUT enzymes, Trm10 does not share the same quaternary structure or key sequences with other members of the SPOUT family, suggesting a novel mechanism of catalysis. To investigate the mechanism of m1G9 methylation by Trm10, we performed a biochemical and kinetic analysis of Trm10 and variants with alterations in highly conserved residues, using crystal structures solved in the absence of tRNA as a guide. Here we demonstrate that a previously proposed general base residue (D210 in Saccharomyces cerevisiae Trm10) is not likely to play this suggested role in the chemistry of methylation. Instead, pH-rate analysis suggests that D210 and other conserved carboxylate-containing residues at the active site collaborate to establish an active site environment that promotes a single ionization that is required for catalysis. Moreover, Trm10 does not depend on a catalytic metal ion, further distinguishing it from the other known SPOUT m1G methyltransferase, TrmD. These results provide evidence for a non-canonical tRNA methyltransferase mechanism that characterizes the Trm10 enzyme family.