How Natural Enzymes and Synthetic Ribozymes Generate Methylated Nucleotides in RNA.

Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S-adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase.

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB.

Among the large and diverse collection of tRNA modifications, 7-methylguanosine (m7G) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNAPhe enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of WT and single substitution variants of TrmB with tRNA. Our results reveal the role of S-adenosylmethionine for rapid and stable tRNA binding, the rate-limiting nature of m7G46 catalysis for tRNA release, and the importance of residues R26, T127, and R155 across the entire surface of TrmB for tRNA binding.

Crosstalk between the tRNA methyltransferase Trm1 and RNA chaperone La influences eukaryotic tRNA maturation.

tRNAs undergo an extensive maturation process involving posttranscriptional modifications often associated with tRNA structural stability and promoting the native fold. Impaired posttranscriptional modification has been linked to human disease, likely through defects in translation, mitochondrial function, and increased susceptibility to degradation by various tRNA decay pathways. More recently, evidence has emerged that bacterial tRNA modification enzymes can act as tRNA chaperones to guide tRNA folding in a manner independent from catalytic activity. Here, we provide evidence that the fission yeast tRNA methyltransferase Trm1, which dimethylates nuclear- and mitochondrial-encoded tRNAs at G26, can also promote tRNA functionality in the absence of catalysis. We show that WT and catalytic-dead Trm1 are active in an in vivo tRNA-mediated suppression assay and possess RNA strand annealing and dissociation activity in vitro, similar to previously characterized RNA chaperones. Trm1 and the RNA chaperone La have previously been proposed to function synergistically in promoting tRNA maturation, yet we surprisingly demonstrate that La binding to nascent pre-tRNAs decreases Trm1 tRNA dimethylation in vivo and in vitro. Collectively, these results support the hypothesis for tRNA modification enzymes that combine catalytic and noncatalytic activities to promote tRNA maturation, as well as expand our understanding of how La function can influence tRNA modification.

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.

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.

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.

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK.

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

Evaluating the druggability of TrmD, a potential antibacterial target, through design and microbiological profiling of a series of potent TrmD inhibitors.

The post-transcriptional modifier tRNA-(N1G37) methyltransferase (TrmD) has been proposed to be essential for growth in many Gram-negative and Gram-positive pathogens, however previously reported inhibitors show only weak antibacterial activity. In this work, optimisation of fragment hits resulted in compounds with low nanomolar TrmD inhibition incorporating features designed to enhance bacterial permeability and covering a range of physicochemical space. The resulting lack of significant antibacterial activity suggests that whilst TrmD is highly ligandable, its essentiality and druggability are called into question.

Trimethylguanosine synthase 1 (Tgs1) is involved in Swi6/HP1-independent siRNA production and establishment of heterochromatin in fission yeast.

In fission yeast, siRNA generated by RNA interference (RNAi) factors plays critical roles in establishment and maintenance of heterochromatin. To achieve efficient siRNA synthesis, RNAi factors assemble on heterochromatin via association with Swi6, a homologue of heterochromatin protein 1 (HP1), and heterochromatic noncoding RNA (hncRNA) retained on chromatin. In addition, spliceosomes formed on hncRNA introns recruit RNAi factors to hncRNA and heterochromatin. Small nuclear RNAs, components of the spliceosome, have a trimethylguanosine (TMG) cap that is generated by Tgs1-dependent hypermethylation of the normal m7G cap; this cap is required for efficient splicing of some mRNAs in budding yeast and Drosophila. In this study, we found that loss of Tgs1 in fission yeast destabilizes centromeric heterochromatin. Tgs1 was required for Swi6-independent siRNA synthesis, as well as for the establishment of centromeric heterochromatin. Loss of Tgs1 affected the splicing efficiency of hncRNA introns in the absence of Swi6. Furthermore, some hncRNAs have a TMG cap, and we found that loss of Tgs1 diminished the chromatin binding of these hncRNAs. Together, these results suggest that the Tgs1-dependent TMG cap plays critical roles in establishment of heterochromatin by ensuring spliceosome-dependent recruitment of RNAi factors and regulating the binding between chromatin and hncRNA.

Structural and functional insights into Archaeoglobus fulgidus m2G10 tRNA methyltransferase Trm11 and its Trm112 activator.

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.

The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain.

tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA's D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA's tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis.

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.

Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites.

S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intriguingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess significantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB and on all-atom molecular dynamics simulations in water). We identified structural descriptors-angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs compact binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water.

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant Kd of TrmB and tRNAPhe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNAPhe complex structure.

Archaeal NSUN6 catalyzes m5C72 modification on a wide-range of specific tRNAs.

Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m5C) at C72 of four specific tRNAs, and its homologs are present only in higher eukaryotes and hyperthermophilic archaea. Archaeal NSun6 homologs possess conserved catalytic residues, but have distinct differences in their RNA recognition motifs from eukaryotic NSun6s. Until now, the biochemical properties and functions of archaeal NSun6 homologs were unknown. In archaeon Pyrococcus horikoshii OT3, the gene encoding the NSun6 homolog is PH1991. We demonstrated that the PH1991 protein could catalyze m5C72 formation on some specific PhtRNAs in vitro and was thus named as PhNSun6. Remarkably, PhNSun6 has a much wider range of tRNA substrates than hNSun6, which was attributed to its tRNA substrate specificity. The mechanism was further elucidated using biochemical and crystallographic experiments. Structurally, the binding pocket for nucleotide 73 in PhNSun6 is specific to accommodate U73 or G73-containing PhtRNAs. Furthermore, PhNSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by PhNSun6. Functionally, the m5C72 modification could slightly promote the thermal stability of PhtRNAs, but did not affect the amino acid accepting activity of PhtRNAs.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Structure of tRNA methyltransferase complex of Trm7 and Trm734 reveals a novel binding interface for tRNA recognition.

The complex between Trm7 and Trm734 (Trm7-Trm734) from Saccharomyces cerevisiae catalyzes 2'-O-methylation at position 34 in tRNA. We report biochemical and structural studies of the Trm7-Trm734 complex. Purified recombinant Trm7-Trm734 preferentially methylates tRNAPhe transcript variants possessing two of three factors (Cm32, m1G37 and pyrimidine34). Therefore, tRNAPhe, tRNATrp and tRNALeu are specifically methylated by Trm7-Trm734. We have solved the crystal structures of the apo and S-adenosyl-L-methionine bound forms of Trm7-Trm734. Small angle X-ray scattering reveals that Trm7-Trm734 exists as a hetero-dimer in solution. Trm7 possesses a Rossmann-fold catalytic domain, while Trm734 consists of three WD40 ß-propeller domains (termed BPA, BPB and BPC). BPA and BPC form a unique V-shaped cleft, which docks to Trm7. The C-terminal region of Trm7 is required for binding to Trm734. The D-arm of substrate tRNA is required for methylation by Trm7-Trm734. If the D-arm in tRNAPhe is docked onto the positively charged area of BPB in Trm734, the anticodon-loop is located near the catalytic pocket of Trm7. This model suggests that Trm734 is required for correct positioning of tRNA for methylation. Additionally, a point-mutation in Trm7, which is observed in FTSJ1 (human Trm7 ortholog) of nosyndromic X-linked intellectual disability patients, decreases the methylation activity.