Distinct determinants of tRNA recognition by the TrmD and Trm5 methyl transferases

TrmD and Trm5 are, respectively, the bacterial and eukarya/archaea methyl transferases that catalyze transfer of the methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 in tRNA to synthesize m1G37-tRNA. The m1G37 modification prevents tRNA frameshifts on the ribosome by assuring correct codon-anticodon pairings, and thus is essential for the fidelity of protein synthesis. Although TrmD and Trm5 are derived from unrelated AdoMet families and recognize the cofactor using distinct motifs, the question of whether they select G37 on tRNA by the same, or different, mechanism has not been answered. Here we address this question by kinetic analysis of tRNA truncation mutants that lack domains typically present in the canonical L shaped structure, and by evaluation of the site of modification on tRNA variants with an expanded or contracted anticodon loop. With both experimental approaches, we show that TrmD and Trm5 exhibit separate and distinct mode of tRNA recognition, suggesting that they evolved by independent and non-overlapping pathways from their unrelated AdoMet families. Our results also shed new light onto the significance of the m1G37 modification in the controversial quadruplet-pairing model of tRNA frameshift suppressors.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

Conservation of structure and mechanism by Trm5 enzymes

Enzymes of the Trm5 family catalyze methyl transfer from S-adenosyl methionine (AdoMet) to the N¹ of G37 to synthesize m¹G37-tRNA as a critical determinant to prevent ribosome frameshift errors. Trm5 is specific to eukaryotes and archaea, and it is unrelated in evolution from the bacterial counterpart TrmD, which is a leading anti-bacterial target. The successful targeting of TrmD requires detailed information on Trm5 to avoid cross-species inhibition. However, most information on Trm5 is derived from studies of the archaeal enzyme Methanococcus jannaschii (MjTrm5), whereas little information is available for eukaryotic enzymes. Here we use human Trm5 (Homo sapiens; HsTrm5) as an example of eukaryotic enzymes and demonstrate that it has retained key features of catalytic properties of the archaeal MjTrm5, including the involvement of a general base to mediate one proton transfer. We also address the protease sensitivity of the human enzyme upon expression in bacteria. Using the tRNA-bound crystal structure of the archaeal enzyme as a model, we have identified a single substitution in the human enzyme that improves resistance to proteolysis. These results establish conservation in both the catalytic mechanism and overall structure of Trm5 between evolutionarily distant eukaryotic and archaeal species and validate the crystal structure of the archaeal enzyme as a useful model for studies of the human enzyme.     

Control of Catalytic Cycle by a Pair of Analogous tRNA Modification Enzymes

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here, we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3′ position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor. TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinity to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed half-of-the-sites reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel with those between the two classes of aminoacyl-tRNA synthetases, which use distinct active site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.     

Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition

tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to G(37) within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G(36)G(37) in the active site of TrmD and the catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD(2)-tRNA(2) complex, which provides insights into recognition of the general tRNA structure by TrmD.     

Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Delta trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA(Met)(f)). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA(Met)(f) and tRNA(Phe). High pressure liquid chromatography analysis showed the methylated product to be m(1)G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1-33 (Delta1-33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Delta1-33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m(1)G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.     

Exploring GpG bases next to anticodon in tRNA subsets

Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes. Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37^th position and it is next to the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m¹G37) methyltransferase (TrmD). It is deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37^th and 38^th position except three tRNA subsets having G residues at 36^th and 39^th positions. Therefore we propose that m1G37 modification may be feasible at 36^th, 37^th, 38^th, 39^th and 40^th positions next to the anticodon of tRNAs. Collectively, methylation at G residues close to the anticodon may be possible at different positions and without restriction of anticodon 3^rd base A, C, U or G.     

Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.     

Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline

The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the kcat of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the Km and little effect on the Kd of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.     

Distinct origins of tRNA(m1G37) methyltransferase

The enzyme tRNA(m1G37) methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the N1 position of G37 in the anticodon loop of a subset of tRNA. The modified guanosine is 3' to the anticodon and is important for maintenance of reading frame during decoding of genetic information. While the methyltransferase is well conserved in bacteria and is easily identified (encoded by the trmD gene), the identity of the enzyme in eukarya and archaea is less clear. Here, we report that the enzyme encoded by Mj0883 of Methanocaldococcus jannaschii is the archaeal counterpart of the bacterial TrmD. However, despite catalyzing the same reaction and displaying similar enzymatic properties, MJ0883 and bacterial TrmD are completely unrelated in sequence. The catalytic domain of MJ0883, when aligned with the five known structural folds (I-V) that have been described to bind AdoMet, is of the class I fold, similar to the ancient Rossmann fold that binds nucleotides. In contrast, the catalytic domain of the bacterial TrmD has the unusual class IV fold of a trefoil knot structure. Thus, both the sequence and structural arrangements of tRNA(m1G37) methyltransferase have distinct evolutionary origins among primary kingdoms, revealing an unexpected but remarkable non-orthologous gene displacement to achieve an important tRNA modification.     

Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.     

Trm11p and Trm112p are both required for the formation of 2-methylguanosine at position 10 in yeast tRNA

N(2)-Monomethylguanosine-10 (m(2)G10) and N(2),N(2)-dimethylguanosine-26 (m(2)(2)G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m(2)(2)G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m(2)G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m(2)G10 and m(2)(2)G26 affects tRNA metabolism or functioning.     

Crystal structure of archaeal tRNA(m(1)G37)methyltransferase aTrm5

Methylation of the N1 atom of guanosine at position 37 in tRNA, the position 3'-adjacent to the anticodon, generates the modified nucleoside m(1)G37. In archaea and eukaryotes, m(1)G37 synthesis is catalyzed by tRNA(m(1)G37)methyltransferase (archaeal or eukaryotic Trm5, a/eTrm5). Here we report the crystal structure of archaeal Trm5 (aTrm5) from Methanocaldococcus jannaschii (formerly known as Methanococcus jannaschii) in complex with the methyl donor analogue at 2.2 A resolution. The crystal structure revealed that the entire protein is composed of three structural domains, D1, D2, and D3. In the a/eTrm5 primary structures, D2 and D3 are highly conserved, while D1 is not conserved. The D3 structure is the Rossmann fold, which is the hallmark of the canonical class-I methyltransferases. The a/eTrm5-defining domain, D2, exhibits structural similarity to some class-I methyltransferases. In contrast, a DALI search with the D1 structure yielded no structural homologues. In the crystal structure, D3 contacts both D1 and D2. The residues involved in the D1:D3 interactions are not conserved, while those participating in the D2:D3 interactions are well conserved. D1 and D2 do not contact each other, and the linker between them is disordered. aTrm5 fragments corresponding to the D1 and D2-D3 regions were prepared in a soluble form. The NMR analysis of the D1 fragment revealed that D1 is well folded by itself, and it did not interact with either the D2-D3 fragment or the tRNA. The NMR analysis of the D2-D3 fragment revealed that it is well folded, independently of D1, and that it interacts with tRNA. Furthermore, the D2-D3 fragment was as active as the full-length enzyme for tRNA methylation. The positive charges on the surface of D2-D3 may be involved in tRNA binding. Therefore, these findings suggest that the interaction between D1 and D3 is not persistent, and that the D2-D3 region plays the major role in tRNA methylation.     

Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.     

Crystal Structure of Methanocaldococcus jannaschii Trm4 Complexed with Sinefungin

tRNA:m⁵C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 Å and 2.3 Å resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix α8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.     

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m¹G₉ modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m¹G₉ in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m¹G₉ methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m¹G₉ containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.     

Ligand-mediated anticodon conformational changes occur during tRNA methylation by a TrmD methyltransferase

Orthologs of TrmD, G37 tRNA methyltransferases, have been analyzed with regard to post-tRNA binding events required to move the residue G37 in proximity to bound AdoMet for catalysis. This was approached initially by probing tRNA with T2 nuclease or Pb acetate in the presence, then absence, of Escherichia coli TrmD protein. Cleavage patterns clearly show that portions of the anticodon loop phosphodiester backbone are protected from cleavage only in the presence of sinefungin, a potent AdoMet analogue. This demonstrates that there must be considerable movement of the loop region and/or protein as the AdoMet site is occupied. Florescence energy transfer experiments were employed to better assess the movement of the G37 and G36 base residues in response to occupancy of the AdoMet site. When the Streptococcus pneumoniae TrmD protein was bound to synthetic tRNA(1)(Leu) substituted with 2-aminopurine at positions 36 and 37, fluorescence energy transfer analysis showed that a decrease in 2-aminopurine fluorescence occurs only when AdoMet is present. Taken together, these results suggest that the base to be methylated by the TrmD protein is mobilized into the active center after tRNA binding only when the AdoMet site is occupied.