N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of ∼150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80°C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.
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.
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.
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.
The N1-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m1A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.
AdoMet; tRNA modification enzyme; Methylation; X-ray crystal structure; Structural genomics
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.
N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
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.
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.
Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT.
As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases.
Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).
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.
We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA(CAA)Leu. Using transcripts of intron-less and intron-containing human tRNA(CAA)Leu genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNALeu transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of human pre-tRNA(CAA)Leu and identification of human gene encoding tRNA methylase responsible for this reaction.
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.
Modified nucleosides in tRNAs play important roles in tRNA structure, biosynthesis and function, and serve as crucial determinants of bacterial growth and virulence. In the yeast Saccharomyces cerevisiae, mutants defective in N1-methylation of a highly conserved adenosine (A58) in the TΨC loop of initiator tRNA are non-viable. The yeast m1A58 methyltransferase is a heterotetramer consisting of two different polypeptide chains, Gcd14p and Gcd10p. Interestingly, while m1A58 is not found in most eubacteria, the mycobacterial tRNAs have m1A58. Here, we report on the cloning, overexpression, purification and biochemical characterization of the Rv2118c gene-encoded protein (Rv2118p) from Mycobacterium tuberculosis, which is homologous to yeast Gcd14p. We show that Rv2118c codes for a protein of ∼31 kDa. Activity assays, modified base analysis and primer extension experiments using reverse transcriptase reveal that Rv2118p is an S-adenosyl-l-methionine-dependent methyltransferase which carries out m1A58 modification in tRNAs, both in vivo and in vitro. Remarkably, when expressed in Escherichia coli, the enzyme methylates the endogenous E.coli initiator tRNA essentially quantitatively. Furthermore, unlike its eukaryotic counterpart, which is a heterotetramer, the mycobacterial enzyme is a homotetramer. Also, the presence of rT modification at position 54, which was found to inhibit the Tetrahymena pyriformis enzyme, does not affect the activity of Rv2118p. Thus, the mycobacterial m1A58 tRNA methyltransferase possesses distinct biochemical properties. We discuss aspects of the biological relevance of Rv2118p in M.tuberculosis, and its potential use as a drug target to control the growth of mycobacteria.
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m5U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N5, N10-methylenetetrahydrofolate (CH2H4folate). We have identified the gene for Bacillus subtilis tRNA:m5U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor.
The Escherichia coli trmA gene encodes the tRNA(m5U54)methyltransferase, which catalyses the formation of m5U54 in tRNA. During the synthesis of m5U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m5U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m5U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m5U in position 1939 of E. coli 23 S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m5U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.
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.
The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA m1G37 methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, m1G37 modification was reported to take place on three conserved tRNA subsets (tRNAArg, tRNALeu, tRNAPro); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the m1G37 modification. The present study reveals Gm18, m1G37 modification, and positions of m1G that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the m1G and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs (tRNAMet, tRNAPro, tRNAVal). Whereas the m1G37 modification base G is formed only on tRNAArg, tRNALeu, tRNAPro, and tRNAHis, the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and m1G modification occur irrespective of a G residue in tRNAs.
anticodon; Gm18 and m1G modification sites; tRNA subsets
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.
Trm5; TrmD; burst kinetics; tRNA(m1G37); half-of-the-site reactivity
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.
Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2 -dimethylguanosine (m22G26). In Saccharomyces cerevisiae , this reaction is catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase. As a prerequisite for future studies, the yeast TRM1 gene was expressed in Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively purified. rTrm1p catalysed both the mono- and dimethylation of G26 in vivo in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA. The TRM1 gene from two independent wild-type yeast strains differed at 14 base positions causing two amino acid exchanges . Exchange of the original Ser467 for Leu caused a complete loss of enzyme activity in vitro against trm1 yeast tRNA. Comparatively short N- or C-terminal deletions from the 570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme activity, as did some point mutations within these regions. This indicated that the protein is not a two domain peptide with the enzyme activity localised to one of the domains, but rather that both ends of the polypeptide seem to interact to influence the conformation of those parts that make up the RNA-binding site and/or the active site of the enzyme.
Bacterial transfer RNA can suppress the immunostimulatory activity of other bacterial tRNAs as a result of the presence of a guanosine modification.
Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2′-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus–infected PBMCs.
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.
The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 126.96.36.199) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF. Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa). The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.
Up to now, Ψ formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Ψ35 formation in pre-tRNATyr(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Ψ-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3′ to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Ψ35 in tRNATyr(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNATyr(GUA) abolished Ψ35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNATyr did not affect Ψ35 formation in this RNA when using an S. solfataricus extract.
Transfer RNAs are synthesized as a primary transcript that is processed to
produce a mature tRNA. As part of the maturation process, a subset of the
nucleosides are modified. Modifications in the anticodon region often
modulate the decoding ability of the tRNA. At position 34, the majority of
yeast cytosolic tRNA species that have a uridine are modified to
5-methoxycarbonylmethyl-uridine (mcm5U) or
5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The
formation of mcm5 and ncm5 side chains involves a
complex pathway, where the last step in formation of mcm5 is a
methyl esterification of cm5 dependent on the Trm9 and Trm112
Methodology and Principal Findings
Both Trm9 and Trm112 are required for the last step in formation of
mcm5 side chains at wobble uridines. By co-expressing a
histidine-tagged Trm9p together with a native Trm112p in E.
coli, these two proteins purified as a complex. The presence of
Trm112p dramatically improves the methyltransferase activity of Trm9p
in vitro. Single tRNA species that normally contain
mcm5U or mcm5s2U nucleosides were
isolated from trm9Δ or trm112Δ
mutants and the presence of modified nucleosides was analyzed by HPLC. In
both mutants, mcm5U and mcm5s2U nucleosides
are absent in tRNAs and the major intermediates accumulating were
ncm5U and ncm5s2U, not the expected
cm5U and cm5s2U.
Trm9p and Trm112p function together at the final step in formation of
mcm5U in tRNA by using the intermediate cm5U as a
substrate. In tRNA isolated from trm9Δ and
trm112Δ strains, ncm5U and
ncm5s2U nucleosides accumulate, questioning the
order of nucleoside intermediate formation of the mcm5 side
chain. We propose two alternative explanations for this observation. One is
that the intermediate cm5U is generated from ncm5U by
a yet unknown mechanism and the other is that cm5U is formed
before ncm5U and mcm5U.