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1.  Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli  
tRNA (m7G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution.
Transfer RNA (tRNA) (m7G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7-­methylguanosine (m7G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21.
doi:10.1107/S1744309108020241
PMCID: PMC2494960  PMID: 18678947
tRNA (m7G46) methyltransferase; Rossmann-fold methyltransferase family
2.  Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein 
Nucleic Acids Research  2011;39(14):6249-6259.
Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.
doi:10.1093/nar/gkr176
PMCID: PMC3152332  PMID: 21478168
3.  Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate 
Nucleic Acids Research  2013;42(1):509-525.
Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N1 methylation of guanine at Position 9 (m1G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.
doi:10.1093/nar/gkt869
PMCID: PMC3874184  PMID: 24081582
4.  The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL 
Nucleic Acids Research  2013;41(16):7828-7842.
Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.
doi:10.1093/nar/gkt568
PMCID: PMC3763551  PMID: 23804755
5.  Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life 
Nucleic Acids Research  2012;40(11):5149-5161.
Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNAPhe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m2G6 (N2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. TTCTrmN and PfTrm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNAPhe of T. thermophilus and via site-directed mutagenesis.
doi:10.1093/nar/gks163
PMCID: PMC3367198  PMID: 22362751
6.  Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions 
Background
tRNA m1A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.
Results
In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.
Conclusions
The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.
doi:10.1186/1472-6807-11-48
PMCID: PMC3281791  PMID: 22168821
7.  Crystallization and preliminary X-ray diffraction crystallographic study of tRNA m1A58 methyltransferase from Saccharomyces cerevisiae  
The expression, purification, crystallization and X-ray diffraction analysis of tRNA m1A58 methyltransferase from S. cerevisiae are reported.
In Saccharomyces cerevisiae, TRM6 and TRM61 compose a tRNA methyltrans­ferase which catalyzes the methylation of the N1 of adenine at position 58 in tRNAs, especially initiator methionine tRNA. TRM61 is the subunit that binds S-­adenosyl-l-methionine and both subunits contribute to target tRNA binding. In order to elucidate the catalytic mechanism of TRM6–TRM61 and the mode of interaction between the two subunits, expression, purification, crystallization and X-­ray diffraction analysis of the TRM6–TRM61 complex were performed in this study. The crystals diffracted to 2.80 Å resolution and belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 139.14, c = 101.62 Å.
doi:10.1107/S174430911103733X
PMCID: PMC3212473  PMID: 22102254
tRNA m1A58 methyltransferase; Saccharomyces cerevisiae
8.  The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli. 
Journal of Bacteriology  1991;173(5):1757-1764.
The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.
Images
PMCID: PMC207327  PMID: 1999392
9.  Control of Catalytic Cycle by A Pair of Analogous tRNA Modification Enzymes 
Journal of molecular biology  2010;400(2):204-217.
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. While both 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 affinities 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 to 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.
doi:10.1016/j.jmb.2010.05.003
PMCID: PMC2892103  PMID: 20452364
Trm5; TrmD; burst kinetics; tRNA(m1G37); half-of-the-site reactivity
10.  Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase 
Nucleic Acids Research  2010;38(18):6206-6218.
The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence.
doi:10.1093/nar/gkq381
PMCID: PMC2952851  PMID: 20483913
11.  N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network 
Nucleic Acids Research  2009;38(3):942-957.
N7-methylguanine at position 46 (m7G46) in tRNA is produced by tRNA (m7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m7G46 modification in tRNAPhe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m1G37, suggesting that the m7G46 positively affects their formations. Although the lack of the m7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNAPhe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNAPhe and tRNAIle. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m7G46 modification supports introduction of other modifications.
doi:10.1093/nar/gkp1059
PMCID: PMC2817472  PMID: 19934251
12.  Genetic organization and transcription from the gene (trmA) responsible for synthesis of tRNA (uracil-5)-methyltransferase by Escherichia coli. 
Journal of Bacteriology  1985;164(3):1117-1123.
The enzyme catalyzing the formation of 5-methyluridine (ribothymidine) in tRNA of Escherichia coli is tRNA (uracil-5)-methyltransferase (EC 2.1.1.35). A 2.8-kilobase EcoRI chromosomal DNA fragment contains trmA, the structural gene for this enzyme. Subcloning, transcription in vitro, Tn5 insertion mutagenesis, and transcriptional fusion experiments were performed to establish the gene organization of the trmA region on the E. coli chromosome. trmA is a monocistronic operon. The trmA promoter was localized by in vitro experiments, and the direction of transcription was shown to be counterclockwise on the standard E. coli K-12 chromosomal map. The level of transcription of trmA in vitro and the expression of protein in minicells equal those of the bla gene of plasmid pBR322.
Images
PMCID: PMC219305  PMID: 2999071
13.  Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding 
Nucleic Acids Research  2007;35(20):6808-6819.
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNAiMet. The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNAiMet, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.
doi:10.1093/nar/gkm574
PMCID: PMC2175304  PMID: 17932071
14.  The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits 
Molecular Biology of the Cell  2012;23(21):4313-4322.
This study shows that Trm112 interacts with and is required for the presence of 18S rRNA methyltransferase Bud23. Also shown is the involvement of Trm112 in 60S biogenesis, thus extending the known functions of Trm112 from tRNA and translation factor methylation to roles in biogenesis of both ribosomal subunits.
We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.
doi:10.1091/mbc.E12-05-0370
PMCID: PMC3484107  PMID: 22956767
15.  MECHANISM AND A PEPTIDE MOTIF FOR TARGETING PERIPHERAL PROTEINS TO THE YEAST INNER NUCLEAR MEMBRANE 
Traffic (Copenhagen, Denmark)  2009;10(9):1243-1256.
Trm1 is a tRNA specific m22G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae. In nuclei Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of <20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (1) translation in the cytoplasm; (2) Ran-dependent import into the nucleoplasm; and (3) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.
doi:10.1111/j.1600-0854.2009.00956.x
PMCID: PMC2788508  PMID: 19602197
inner nuclear membrane; nucleus organization; targeting motif; mitochondrial location
16.  CATALYSIS BY THE SECOND CLASS OF tRNA(m1G37) METHYL TRANSFERASE REQUIRES A CONSERVED PROLINE 
Biochemistry  2006;45(24):7463-7473.
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 kcat of the methylation reaction in steady-state kinetic analysis, and kchem in single turnover kinetic analysis. However, substitution of P267 has milder effect on Km and little effect on 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.
doi:10.1021/bi0602314
PMCID: PMC2517134  PMID: 16768442
17.  The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity 
Nucleic Acids Research  2008;36(15):4929-4940.
The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis.
doi:10.1093/nar/gkn437
PMCID: PMC2528175  PMID: 18653523
18.  Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth 
Journal of Bacteriology  2004;186(8):2346-2354.
Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathit{tRNA}}_{{\mathit{CAG}}}^{{\mathit{Leu}}}\end{equation*}\end{document}. Other heterologous nonsubstrate tRNA species, like \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathit{tRNA}}_{{\mathit{GGT}}}^{{\mathit{Thr}}}\end{equation*}\end{document}, tRNAPhe, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathit{tRNA}}_{{\mathit{TGC}}}^{{\mathit{Ala}}}\end{equation*}\end{document}, bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).
doi:10.1128/JB.186.8.2346-2354.2004
PMCID: PMC412112  PMID: 15060037
19.  Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications 
RNA Biology  2012;9(7):990-1001.
Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm5U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), classified as key determinants of translational fidelity. We also show that in the absence of mcm5U and mcm5s2U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways.
doi:10.4161/rna.20531
PMCID: PMC3495739  PMID: 22832247
Trm9; codon; heat shock response; modification; protein stress; tRNA; translation infidelity; unfolded protein response; wobble base
20.  Distinct Determinants of tRNA Recognition by the TrmD and Trm5 Methyl Transferases 
Journal of molecular biology  2007;373(3):623-632.
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.
doi:10.1016/j.jmb.2007.08.010
PMCID: PMC2064070  PMID: 17868690
tRNA(m1G37) methyl transferase; anticodon stem-loop; frameshift suppressor tRNA; m1G37
21.  Transfer RNA Methytransferases and Their Corresponding Modifications in Budding Yeast and Humans: Activities, Predications, and Potential Roles in Human Health 
DNA and Cell Biology  2012;31(4):434-454.
Throughout the kingdoms of life, transfer RNA (tRNA) undergoes over 100 enzyme-catalyzed, methyl-based modifications. Although a majority of the methylations are conserved from bacteria to mammals, the functions of a number of these modifications are unknown. Many of the proteins responsible for tRNA methylation, named tRNA methyltransferases (Trms), have been characterized in Saccharomyces cerevisiae. In contrast, only a few human Trms have been characterized. A BLAST search for human homologs of each S. cerevisiae Trm revealed a total of 34 human proteins matching our search criteria for an S. cerevisiae Trm homolog candidate. We have compiled a database cataloging basic information about each human and yeast Trm. Every S. cerevisiae Trm has at least one human homolog, while several Trms have multiple candidates. A search of cancer cell versus normal cell mRNA expression studies submitted to Oncomine found that 30 of the homolog genes display a significant change in mRNA expression levels in at least one data set. While 6 of the 34 human homolog candidates have confirmed tRNA methylation activity, the other candidates remain uncharacterized. We believe that our database will serve as a resource for investigating the role of human Trms in cellular stress signaling.
doi:10.1089/dna.2011.1437
PMCID: PMC3322404  PMID: 22191691
22.  Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14 
Nucleic Acids Research  2011;39(17):7641-7655.
The modified nucleosides N2-methylguanosine and N22-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m2G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m2G at position 6 in tRNACys. The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m2G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20–30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m2G6 tRNA modification.
doi:10.1093/nar/gkr475
PMCID: PMC3177210  PMID: 21693558
23.  New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA 
Nucleic Acids Research  2010;38(19):6533-6543.
Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N1-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N1 atom of a purine residue is discussed.
doi:10.1093/nar/gkq451
PMCID: PMC2965216  PMID: 20525789
24.  Identifying the methyltransferases for m5U747 and m5U1939 in 23S rRNA using MALDI mass spectrometry 
Nucleic Acids Research  2003;31(16):4738-4746.
There are three sites of m5U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF+ and YbjF– strains showed that the latter differed only in the lack of the m5U747 modification. With this report, the functions of all the E.coli m5U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).
PMCID: PMC169892  PMID: 12907714
25.  Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575 
Molecular and Cellular Biology  2012;32(12):2254-2267.
Posttranscriptional and posttranslational modification of macromolecules is known to fine-tune their functions. Trm112 is unique, acting as an activator of both tRNA and protein methyltransferases. Here we report that in Saccharomyces cerevisiae, Trm112 is required for efficient ribosome synthesis and progression through mitosis. Trm112 copurifies with pre-rRNAs and with multiple ribosome synthesis trans-acting factors, including the 18S rRNA methyltransferase Bud23. Consistent with the known mechanisms of activation of methyltransferases by Trm112, we found that Trm112 interacts directly with Bud23 in vitro and that it is required for its stability in vivo. Consequently, trm112Δ cells are deficient for Bud23-mediated 18S rRNA methylation at position G1575 and for small ribosome subunit formation. Bud23 failure to bind nascent preribosomes activates a nucleolar surveillance pathway involving the TRAMP complexes, leading to preribosome degradation. Trm112 is thus active in rRNA, tRNA, and translation factor modification, ideally placing it at the interface between ribosome synthesis and function.
doi:10.1128/MCB.06623-11
PMCID: PMC3372273  PMID: 22493060

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