Distinct Origins of tRNA(m1G37) Methyltransferase

Distinct Origins of tRNA(m1G37) Methyltransferase

Thomas Christian, Caryn Evilia, Sandra Williams and Ya-Ming Hou*

Department of Biochemistryand Molecular PharmacologyThomas Jefferson University233 South 10th Street, BLSB220, Philadelphia, PA 19107USA

The enzyme tRNA(m1G37) methyltransferase catalyzes the transfer of amethyl group from S-adenosyl-L-methionine (AdoMet) to the N1 positionof G37 in the anticodon loop of a subset of tRNA. The modified guanosineis 30 to the anticodon and is important for maintenance of reading frameduring decoding of genetic information. While the methyltransferase iswell conserved in bacteria and is easily identified (encoded by the trmDgene), the identity of the enzyme in eukarya and archaea is less clear.Here, we report that the enzyme encoded by Mj0883 of Methanocaldococcusjannaschii is the archaeal counterpart of the bacterial TrmD. However,despite catalyzing the same reaction and displaying similar enzymaticproperties, MJ0883 and bacterial TrmD are completely unrelated insequence. The catalytic domain of MJ0883, when aligned with the fiveknown structural folds (I–V) that have been described to bind AdoMet,is of the class I fold, similar to the ancient Rossmann fold that bindsnucleotides. In contrast, the catalytic domain of the bacterial TrmD hasthe unusual class IV fold of a trefoil knot structure. Thus, both thesequence and structural arrangements of tRNA(m1G37) methyltransferasehave distinct evolutionary origins among primary kingdoms, revealingan unexpected but remarkable non-orthologous gene displacement toachieve an important tRNA modification.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: methylation; 1-methylguanosine; AdoMet; tRNA maturation*Corresponding author

Introduction

All naturally occurring tRNA molecules containbase or backbone modifications that impart a richdiversity to structures and functions not observedin other RNAs. In particular, position 37 (30 to theanticodon) contains one of the largest variety ofmodifications.1,2 For tRNAs that contain G37, suchas those reading codons of CYN or CGG (i.e.specific for leucine, proline, and arginine), themodification is invariably 1-methylguanosine(m1G).1 This modification is conserved acrossbroad phylogenetic boundaries and is present inthe organism of the smallest genome (Mycoplasmagenitalium), and in organelles, such as mito-chondria and chloroplasts, where the codon usageis different from that of the “universal” geneticcode.3 The m1G37 modification is essential formaintaining the reading frame fidelity.4 The

absence of this modification results in elevatederror rates of þ1 frame-shifting,3,5 – 7 relaxed amino-acyl-tRNA selection at the ribosome,8 reduced rateof polypeptide elongation,9 and decreased speci-ficity of tRNA aminoacylation.10 The enzymeresponsible for the m1G37 modification istRNA(m1G37)methyl transferase (MTase), whichis the product of the trmD and trm5 genes in bac-teria and yeast, respectively.3,11 Disruptions intrmD or trm5 lead to severely retarded growthrates, further emphasizing the importance of them1G37 modification.

The bacterial TrmDs are highly similar, sharing33% identity and 75% similarity amongthemselves,5 and thus are easily identified fromgenomic databases. Recently, three crystal struc-tures of TrmD have been solved, one each fromHaemophilus influenzae,12 Escherichia coli,13 andAquifex aeolicus,14 which confirmed the sequenceand structural similarity of these enzymes. Thestructure of TrmD is divided into an N-terminaldomain that binds the AdoMet cofactor, a C-termi-nal domain that presumably binds the targettRNA, and a flexible linker between the twodomains. The AdoMet-binding domain has anunusual trefoil knot structure not seen in the

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: AdoMet, S-adenosyl-L-methionine; m1G, 1-methylguanosine; MTase, methyltransferase; 2D-TLC, two-dimensional thin layerchromatography; IPTG, isopropyl-thio b-D-galactoside.

doi:10.1016/j.jmb.2004.04.025 J. Mol. Biol. (2004) 339, 707–719

common AdoMet folds present in the vast majorityof MTases.15 – 17 Most notably, TrmD is a homo-dimer, in which the AdoMet-binding site of eachmonomer is joined to create a functional catalyticcenter. TrmD is structurally related to the SpoUMTases,18,19 which include the E. coli TrmH thatmethylates tRNA G18 and RlmB 23 S rRNAMTase and the Thermus thermophilis RrmA.20,21

These proteins share the knotted structure forAdoMet-binding and collectively form the SPOUTfamily.19

The similarity between the E. coli TrmD andyeast Sacharomyces cerevisiae Trm5 is significantlyreduced, showing 18% identity and 45% similarity.3

However, despite the presence of the m1G modifi-cation in archaeal tRNA, BLAST searches ofMethanocaldococcus jannaschii, the first sequencedarchaeal genome, yielded no obvious homologs ofthe E. coli TrmD (Y.-M.H., unpublished results).It was only until recently that a gene from thechromosomal bank of the related M. vannielii was

found to rescue a temperature-sensitive (ts) trmDmutant of Salmonella typhimurium.3 The homologof this gene was identified from the genome ofM. jannaschii, which was Mj0883. Intriguingly,while Mj0883 was predicted to encode an RNAmethyl transferase,22 the sequence of this proteinappears completely unrelated to that of E. coliTrmD. The limited similarity raises the question ofthe identity of Mj0883 and whether it is an homolo-gous gene but marked by a high substitution rateor it is unrelated to the bacterial trmD.

To address the biochemical identity of Mj0883,we have cloned and expressed the gene in E. coliand used the purified enzyme to test its substrate-specificity and kinetic property. The enzymeindeed catalyzes the AdoMet-dependent methyltransfer to G37 of several archaeal tRNAs anddisplays many features characteristic of bacterialTrmD. Thus, despite considerable sequence diver-gence, the basic biochemical function is preserved.As an MTase, the sequence of MJ0883 has been

Figure 1. (A) Sequence and cloverleaf structures of M. jannaschii tRNAPro and tRNACys. (B) The MTase activityof MJ0883 on tRNAPro and tRNACys, and inactivation of the activity by the A37, C37, and U37 mutations in tRNACys.(C) Steady-state kinetic parameters of MJ0883 and E. coli TrmD with respect to the AdoMet cofactor and tRNA sub-strate. Data of E. coli TrmD were from Elkins et al.13

708 tRNA(m1G37) Methyl Transferase

aligned with the five known structural folds (I–V)of AdoMet-dependent MTases in databases.23 Thisalignment reveals a closer relationship betweenMJ0883 and eukaryotic Trm5 and groups themtogether with the class I MTases that have themost common AdoMet fold related to the classicRossmann fold. In contrast, the bacterial TrmDbelongs to class IV. This predicts a radically dif-ferent AdoMet fold of MJ0883/eukaryotic Trm5from that of bacterial TrmD, which is unexpectedgiven the general importance of the tRNA(m1G37)modification. The possibility of distinct AdoMetfolds offers new perspectives for the design of anti-biotics that can specifically target bacterial TrmD,and provides important evolutionary insights intothe divergence of tRNA(m1G37) MTase.

Results and Discussion

Methyl transfer to tRNA(G37) by MJ0883

The protein MJ0883 (336 amino acid residuesfrom the open reading frame with the addition ofa C-terminal His-tag) was expressed in E. coli andpurified to homogeneity by a heat treatment (dueto the thermal stability of M. jannaschii), followedby binding to a metal-affinity resin, and by cationexchange on FPLC. The purified protein showedan apparent molecular mass of 42 kDa on SDS-PAGE. To test the MTase activity, the unmodifiedtranscripts of M. jannaschii tRNAPro/UGG andtRNACys/GCA were chosen as substrates, both ofwhich have G37 (Figure 1(A)). Using [3H]AdoMetas the methyl donor, MJ0883 was assayed for itsability to catalyze transfer of the labeled methylgroup to tRNA, which was acid precipitated on fil-ter pads and quantified. Both tRNAPro and tRNACys

were methylated to ,30% in 30 minutes (Figure1(B)). The partial methylation may be due to thefact that tRNA transcripts were used as substrates,which can be heterogeneous in conformations inthe absence of other modified bases. This is consist-ent with methylation of E. coli tRNALeu by E. coliTrmD under optimized conditions,24 which is also30%. The heterogeneous conformations were alsoreflected in the 30–50% levels of aminoacylationplateau of the tRNAs. Because tRNAPro andtRNACys differ in the sequence context of G37(G36 in tRNAPro, A36 in tRNACys), the comparablemethylation by MJ0883 suggests that both areefficiently recognized. This is different from thebacterial TrmD, which only recognizes G37 inthe context of G36 and can even use the G36G37dinucleotide as a substrate.25 In contrast, the yeastTrm5 has a more relaxed specificity and methylatesG37 in the context of three of the four naturalbases (not U) at position 36.3 To further test thesequence-specificity of MJ0883, several unmodifiedM. jannaschii tRNA transcripts were tested. MJ0883methylated tRNA having A36 (tRNACys/GCA),C36 (tRNAGlu/UUC), and G36 (tRNAArg/UCG,tRNALeu/UAG, tRNAPro/GGG), but not U36

(tRNAArg/UCU), showing the same specificity asthat of yeast Trm5.

To verify that MJ0883 was an enzyme catalyst,the steady-state kinetic parameters of the methyltransfer reaction were determined (Figure 1(C)).The reaction temperature was at 50 8C, whichwas sub-optimal to the growth temperature ofM. jannaschii (83 8C), but was chosen to maintainthe stability of AdoMet. For the AdoMet cofactor,the Vmax and Km parameters of MJ0883 wereclosely similar to those reported for E. coli TrmD.13

Using the transcript of tRNACys as the substrate,MJ0883 also showed comparable Vmax and Km

values to those of E. coli TrmD.13 Thus, despitelimitations of the assay temperature, the overallefficiency of methyl transfer ðVmax=KmÞ of MJ0883was the same or slightly better than that of E. coliTrmD.

To determine that the product of methyl transferby MJ0883 was m1G, the tRNACys transcript wasinternally labeled with [a-32P]GMP, subjected tomethylation, and hydrolyzed by the P1 nuclease togenerate 50 monophosphate nucleosides.25 Thesenucleosides were separated by two-dimensionalthin layer chromatography (2D-TLC) to revealmigration of the labeled guanylate groups. Com-parison of migration of the radioactivity to markernucleotides showed that the methylated transcriptyielded both GMP and m1GMP, whereas theunmethylated control yielded only GMP (Figure2(A) and (B)). The identities of GMP and m1GMPwere confirmed by analysis of the methylated andunmethylated transcripts of tRNALeu generated bythe known tRNA(m1G37) MTase of E. coli TrmD(Figure 2(C) and (D)). Phosphorimaging of guanyl-ate groups showed that 1% of the label in theMJ0883-methylated transcript was localized to

Figure 2. (A) Analysis of GMP and m1GMP by 2D-TLC for MJ0883-catalyzed methylation of M. jannasciitRNACys, versus (B) the unmethylated control. (C) Analy-sis of GMP and m1GMP for the E. coli TrmD-catalyzedmethylation of tRNALeu, versus (D) the unmethylatedcontrol. The origin of the 2D-TLC is circled, and the firstand second dimensions are indicated by arrows.

tRNA(m1G37) Methyl Transferase 709

m1GMP (Figure 2(A)), consistent with one of the24 G residues in tRNACys (Figure 1(A)) that wasmethylated at 30% efficiency under the assay con-dition. No other labeled spots were observed inthe 2D-TLC, suggesting no other modification ofguanylate groups. The same experiment wasrepeated with labeled adenylate, cytidylate, anduridylate in the tRNA transcript, respectively, butrevealed no modified spots (not shown). Thisestablished that MJ0883 was an m1G-specificMTase.

To determine that the site of methylation byMJ0883 was G37, mutant transcripts of tRNACys,each containing A37, C37, or U37 were created

and tested for methylation. All three were inactive,even with elevated levels of MJ0883 (Figure 1(B)),indicating that G37 was necessary for methyl trans-fer. Second, the methylated transcript of tRNACys

was subjected to the nuclease T1 cleavage, whichwas specific to the 30 phosphate of internalG. Because the T1 cleavage requires the nucleaseforming H-bonds with the N1 atom of the guaninebase,26 T1 does not recognize m1G.27 The transcriptof tRNACys was 30 end-labeled by the tRNACCA-adding enzyme, cleaved by T1 under semi-denaturing condition (50 8C, 1 mM EDTA), andthe cleavage fragments were separated by dena-turing PAGE in 7 M urea (Figure 3(A)). The T1

Figure 3. (A) T1 digestion of methylated (þM) and unmethylated (2M) tRNACys of M. jannaschii catalyzed byMJ0883 over a time course of 0, 5, and 15 minutes and examined by a 7 M urea/12% PAGE. Each G residue in tRNACys

is indicated by a dot while several are also shown by their positions. G34 and G37 are marked by arrows, and a parallelT1 analysis of unmethylated E. coli tRNACys is shown to provide the G34 marker. (B) Primer extension of methylated(þM) and unmethylated (2M) tRNACys of M. jannaschii catalyzed by MJ0883. The primer (15-mer) is labeled, andwas incubated with the tRNA at 0.5, 5, and 10 minutes. Extension products are indicated by dots. The positions ofG37, A36, A26, and G15 are indicated by arrows.


cleavage of G34 in the GCA anticodon was easilyidentified, which is one of the most prominentfeatures of the well-characterized E. coli tRNACys

(included as a control.28) Cleavage at all other Gbases was also identified throughout the entiretRNA, indicating completeness of the cleavagereaction. Cleavage at G bases near the very 30 endgenerated fragments too short to be visualized onthe gel. Notably, while cleavage at G37 was promi-nent in the unmethylated tRNA, it was signifi-cantly reduced in the methylated tRNA. Thereduced cleavage at G37, instead of completeelimination, reflected partial methylation byMJ0883. Nonetheless, it was the most apparentcontrast between the methylated and unmethyl-ated transcripts and this contrast was sustainedthroughout the time course of the T1 reaction,suggesting that G37 was the primary site of themethyl transfer.

The site of methylation of G37 was further con-firmed by primer extension, which would beblocked by the presence of m1G, where the methylgroup is at the Watson–Crick face of the base. A15-mer DNA primer was 50 end-labeled, hybri-dized to A57 to C43 of tRNACys over a time course,and was extended by the AMV reverse transcrip-tase. Extension products were separated by 7 Murea/12% (w/v) PAGE and analyzed by auto-radiography (Figure 3(B)). A major extensionblock at G37 was observed in the methylated, butnot the unmethylated, tRNA. This correspondedto synthesis of a 20-mer by extension to A38,providing evidence that the methylation was atG37. A minor extension block at A36 was alsoobserved in the methylated but not the unmethyl-ated tRNA. Although the possibility of methylationat A36 cannot be ruled out, the minor extensionblock most likely resulted from a small fraction ofread-through of m1G37, perhaps by a weak pairingwith the incoming nucleotide. Due to the incom-plete methylation by MJ0883, some primer exten-sion reached the 50 end of the tRNA (G1). BetweenG37 and G1, an extension block at A26 wasobserved in the methylated tRNA, although itwas even more pronounced in the unmethylatedcontrol, suggesting that it was not blocked bymethylation but by the tRNA local secondarystructures that formed during primer extension.Previous studies had identified multiple sequence-dependent local secondary structures in tRNAthat block primer extension.29 The structure-induced termination of primer extension couldalso explain the block at G15 that occurred in themethylated transcript only in prolonged incubationtime.

MJ0883 was also tested by primer extension forits ability to methylate G9 and G26, which areoften modified in tRNA as m1G and (m2)2G,respectively. These two modifications are notpresent in bacteria, but have been identified intotal M. jannaschii tRNA.30 They are present inyeast and are catalyzed by enzymes of distinctgenes (trm10 for m1G9 and trm1 for (m2)2G26).31,32

The sequence of MJ0883 shares a recognizablehomology to the trm1 homologs in archaea. To testmethylation by MJ0883 at G9 or G26, the tRNAPro

transcript, containing both nucleotides, was used(Figure 1(A)). No primer extension block wasdetected at G9 or G26 (not shown), suggestingthat MJ0883 was specific to G37. The m1G37 modi-fication appeared to increase the local rigidity ofthe anticodon stem-loop of tRNACys. This is shownby the decreased secondary structural block ofprimer extension at A26, which is in the anticodonstem (Figure 3(B)). A recent NMR analysis of theanticodon stem-loop of yeast tRNAPhe confirmedthat the m1G37 methylation restricted the con-formational dynamics of the anticodon loop,rendering it less flexible.33

Complementation of a ts trmD mutant ofS. typhimurium

The S. typhimurium mutant GT5454, harboring ats trmD (trmD27, zff-2521 < Tn10 dCm),3 was usedto determine if Mj0883 functionally complementedtrmD. Mj0883 was sub-cloned into the pUC18-derived expression vector pKK223-3, where geneexpression was directly under the control of theIPTG-inducible tac promoter. The parental vectorpUC18 was included as a negative control, whilethe pUMV4 plasmid that encodes the M. vannieliigene, previously shown to complement the defec-tive trmD, was included as a positive control.3

GT5454 strains that harbored each of these plas-mids were induced by IPTG and tested for growthat the non-restrictive temperature (30 8C) andreplica-tested at the restrictive temperature (42 8C)(Figure 4(A)). Clearly, while all three strainsshowed growth at 30 8C, only those expressingMj0883 (pKK-Mj0883) or pUMV4 conferred growthat 42 8C, providing evidence of complementation ofthe ts allele. The growth at 42 8C was monitored atearly stages because trmD is not essential toS. typhimurium and the negative control wouldappear in prolonged incubation. Under the growthassay condition, cells harboring Mj0883 andpUMV4 appeared similar in growth rate, indi-cating functional equivalence of the two genes.

To corroborate the growth complementation, acell lysate of GT5454 expressing Mj0883 was madeand tested for tRNA(m1G37) MTase activity. Theassay used the tRNACys transcript as the substrate,[3H]AdoMet as the cofactor, and was at the semi-restrictive temperature 37 8C so that the control(cells harboring the vector alone) would haveminimal endogenous activity. Indeed, the MTaseactivity was detected in the lysate that expressedMJ0883, and was significantly higher than that ofthe control throughout the assay (Figure 4(B)).Based on the initial rate of methyl transfer, thetRNA(m1G37) MTase activity normalized by mg ofprotein in the MJ0883-containing cell lysate was atleast 35-fold higher than that of the endogenousactivity, which was encoded by the E. coli trmDgene harboring the ts defect. The amount of


overproduction was similar to that of E. coli TrmDexpressed on a multi-copy plasmid in an E. colistrain (17-fold),34 indicating that expression ofMJ0883, as of E. coli TrmD, exerted no adverseeffect in bacteria.

Comparison of biochemical features of MJ0883and TrmD

The strongest similarity between MJ0883 andTrmD was the kinetic property with respect to theAdoMet cofactor and tRNA substrate (Figure1(C)). However, MJ0883 differed from TrmD inoligomeric states. Whereas TrmD is a dimer,MJ0883 existed as a monomer on a gel filtrationcolumn with an elution volume corresponding toan apparent molecular mass of 46 kDa (Figure5(A)). In addition, a gel shift assay was developedto determine the Kd value of MJ0883 to tRNA. Thetranscript of tRNACys was labeled and assayed

with increasing concentrations of MJ0883. The freeand bound tRNA was separated by electrophoresison an agarose gel. Under conditions where theconcentration of the transcript was low (4 nM) rela-tive to the apparent Kd value (300–500 nM; seebelow), the fraction of bound was calculated fromeach binding reaction and plotted against theenzyme concentration according to a hyperbolicbinding relationship. This yielded an apparent Kd

value (enzyme concentration at 50% binding) of,300 nM (Figure 5(B)), which is higher than theKd value of 68 nM for E. coli TrmD as determinedby the plasmon surface resonance.13 When thetRNA was saturating (4 mM), the gel shift assayrevealed a major shift occurring at enzyme concen-trations (1.0–16.0 mM) and a second shift at higherconcentrations (8–36 mM) (Figure 5(C)). The tRNAbinding data were fitted by solving the massconservation equations and by simultaneouslyevaluating the Kd values (Figure 5(D)). This

Figure 4. (A) S. typhimurium GT5454 strains harboring, respectively, the plasmid pUMV4, pUC18, or pKK-Mj0883and their complementation of the ts mutant of trmD at 30 8C and 42 8C. (B) The MTase activity, measured as transferof the [3H]AdoMet to tRNA, of the cell lysate expressing pKK-Mj0883 or pUC18 is based on the same amount ofproteins.


revealed that the first shift represented a 1 : 1 com-plex with a Kd value of 464 nM, similar to thatdetermined for Figure 5(B). Separately, a gel fil-tration on the Suprose 12 sizing column supportedthe 1 : 1 stoichiometry. In the gel filtration (proteincalibrated), the enzyme (20 mM)-tRNA (30 mM)complex migrated as a 79 kDa (Figure 5(A)), whiletRNA alone migrated as 50 kDa. It should benoted that tRNA alone (due to a different densityand shape than those of proteins) did not migrateat the expected molecular mass (25 kDa), but thatthe presence of protein in the complex couldchange the molecular shape of tRNA on gel fil-tration. The 79 kDa value of the complex wasmost consistent with one monomer of enzymebinding to one tRNA. Although there wereadditional shifts detected in the gel shift assay

(Figure 5(C)), these were higher-order complexesthat occurred at higher enzyme concentrations.(For example, curve-fit could identify a 2 : 1enzyme–tRNA complex with a Kd value of1.18 mM; Figure 5(D).) Under physiological con-ditions, where MTases are usually below micro-molar concentrations, the biologically relevantcomplex is therefore the 1 : 1 complex. In contrast,structural modeling of bacterial TrmD has pro-posed a dimer of TrmD binding to two tRNAmolecules.12

Structural modeling of MJ0883 andmutational analysis

To address the question of whether MJ0883 isrelated to TrmD by a high substitution rate that

Figure 5. (A) Gel filtration of MJ0883 and a complex of MJ0883 with tRNACys on the Suprose 12 sizing column, super-imposed with those of molecular mass standards on the same column. (B) Determination of Kd for tRNACys by MJ0883from a gel shift analysis where the tRNA (4 nM) was labeled, and bound to MJ0883, and the fractions of the boundcomplex are plotted against concentrations of MJ0883 to derive an apparent Kd. (C) Determination of the stoichiometryof the MJ0883–tRNA complex by gel shift, showing two forms of bound tRNA. (D) Curve-fit of the stoichiometryanalysis of (C), showing the Kd value of each complex.


masked sequence similarity or is completelyunrelated, a sequence analysis of the protein wasperformed. Using MJ0883 as the query sequence, aPSI/PHI-BLAST search of the NCBI databaseidentified its homologs from the diverse archaealorganisms of methanogens, hyperthermophiles,and extreme halophiles (Figure 6(A)). Interestingly,while homologs of MJ0883 aligned poorly withbacterial TrmDs, they aligned well with eukaryoticTrm5s. To further investigate the MJ0883-likeproteins and their relationship with eukaryoticTrm5s, the entire database of AdoMet-dependentMTases in the current Protein Data Bank (PDB)was searched.15 This database includes .100crystal structures of MTases that can be classifiedinto five distinct families based on the AdoMet-binding fold (classes I–V).23 The general feature ofthese five AdoMet folds is the same, consisting ofalternating b-strands and a-helices, but the struc-tural arrangement of the strands/helices in eachfold is different. Iterative alignments of the Ado-Met fold showed that MJ0883/Trm5 and theirhomologs were most closely related to enzymes ofthe class I fold, which constitute the largest MTasefamily with members ranging widely in substratespecificity (DNA, RNA, protein, small molecule,etc.) and on the targeted atom for methyl transfer

(nitrogen, oxygen, carbon, sulfur, etc.). In the classI family, the closest relative to the MJ0883-likeproteins is the RsmC in bacteria, which is a 16 SrRNA MTase. Remarkably, the RsmC ortholog inM. jannaschii is MJ0882, which is located in thegenome directly preceding MJ0883. The crystalstructure of MJ0882 has been determined as partof the Structural Genomics Initiatives (PDB code1DUS; L. W. Hung et al., unpublished results)15

(Figure 6(A)), and it contains features of the classicRossmann fold of nucleotide-binding and ischaracterized by a seven-stranded b-sheet withthree helices on each side of a bi-lobal aba sand-wich (Figure 6(C)). Several structural elements ofthe class I AdoMet fold17 can be easily inferredfrom the alignment of this protein with theMJ0883-like homologs, as well as the eukaryoticTrm5s (Figure 6(A)).

The alignment was strongly supported by site-directed mutagenesis of MJ0883, where the effectof mutation is evaluated as a percentage of thewild-type activity (Figure 6(B)). For example,homologs of MJ0883/Trm5 have conserved theclass I-specific motif 1 sequence FAGxG at the endof a core b-strand (B3 in MJ0883), where the GxGmotif is known to bind the methionine moiety ofAdoMet, while the hydrophobic Phe binds the

Figure 6. (A) Sequence alignment of the AdoMet domain of homologs of MJ0883 and Trm5, where alternatingb-strands and a-helices of the fold are indicated by arrows and cylinders, respectively. Sequences include those ofM. jannaschii MJ0883 (Mj), M. acetivorans (Ma), M. mazei (Mz), M. thermoautotrophicum (Mth), H. halobium (Hh), andP. furiosus (Pf), and Trm5 of N. crassa (Nc), A. thalania (At), D. melanogaster (Dm), A. gambiae (Ag), S. cerevisiae (Sc), andH. sapiens (Hs). Bottom: alignment of the putative guanine-binding site. (B) Activities of MJ0883 mutants with conser-vative point mutations at sites are indicated by various symbols. All mutant proteins were assayed for steady-stateactivities relative to that of the wild-type. The D201A mutant was 59% active, the G205A/G207A mutant 12.3% active,the D223A mutant 6.2% active, the N265A mutant 11.2% active, and the P267A mutant 1.3% active. (C) Schematicdrawing of the topology of the class I and class IV folds for AdoMet binding, where arrows indicate b-strands andcylinders indicate a-helices. Key regions in each fold responsible for binding the cofactor or catalysis are colored in red.


adenine moiety.16,17,35 Indeed, mutations of the twoGly residues in the GxG tripeptide of MJ0883 elimi-nated nearly 90% of the activity (G205A/G207A,12.3% active relative to the wild-type), confirmingthe importance of the GxG tripeptide. In addition,these homologs have a conserved Asp, four aminoacid residues upstream of the GxG peptide, whichis known to stabilize the stereochemical constraintsof the cofactor.16 Mutation of this Asp showed amoderate activity (D201A in MJ0883; 59% active),suggesting that the stereochemical constraints arenot the major determinant of the activity. Also,these homologs share a Pro as the last residueof the core b-strand B3, which is common to allclass I MTases that target nitrogen (the aminoMTases).17,36 Second, MJ0883/Trm5 homologs haveconserved motifs II and III of the class I fold,which include an acidic residue at the end ofstrand B4 that is known to interact with the ribosehydroxyl groups of AdoMet.35 Mutation of thisacidic residue indeed eliminated more than 90%of the activity (D223A in MJ0883; 6.2% active).This acidic residue is followed by a bulky hydro-phobic side-chain (I224 in MJ0883) that is knownto make van der Waals contact with the AdoMetadenine. They also share an Asn (N225) in the firstposition of the third a-helix (A3), which in class IMTases interacts with the N6 atom of the AdoMetadenine.16 Third, residues immediately prior to theFAGxGx motif in MJ0883/Trm5 have highly hydro-phobic side-chains, which are characteristics of themotif X in the class I MTases. In structurallycharacterized class I enzymes, the motif X residuesare clustered to pack against neighboring b-strandsto form the binding pocket for the AdoMetmethionine.37 Fourth, MJ0883/Trm5 have a con-served NLP motif at the end of the strand B6,which was noted previously22 and is analogous tothe [D/N/S]PP[Y/F] (DPPY) motif in the aminoMTases that target DNA N6-adenine andN4-cytosine.38,39 The DPPY motif in the latter isimportant for catalysis, where the Asp/Asn andthe first Pro serve to properly position the nitrogenatom for nucleophilic attack of the methyl group ofAdoMet.17,40 In MJ0883, mutation of Asn in theNLP motif reduced the activity by almost tenfold(N265A; 11.2% active), while mutation of Proessentially abolished the activity (P267A; 1.3%active). This confirmed the catalytic importance ofthe NLP motif for MJ0883.

In contrast, the trefoil knot structure in bacterialTrmD that binds AdoMet is of the class IV fold,as revealed in the three crystal structures.12 – 14

Whereas the b-sheet in the class I fold follows thetopology of 6 " 7 # 5 " 4 " 1 " 2 " 3 " , that of the classIV fold follows the topology of 7 " 5 " 6 " 1 " 2 " 3 " ,where the first three strands form half of theRossmann fold, while the last three strands(B5–B7) and helices (A4–A6) form the trefoil knot(Figure 6(C)). Although TrmD differs from the con-ventional class IV fold by directing the third strandaway from the core b-sheet, the knot structure isclearly visible, which contains conserved sequence

motifs and provides the binding site for AdoMet.Notably, the cofactor bound to the knotted struc-ture is in a bent conformation,12,13,41 instead of theextended conformation as in the class I fold. In thehomo-dimer of TrmD, mutational analysis hasshown that the active site cleft is formed at thedimer interface, involving residues from the Ado-Met fold of both monomers and also residues inthe flexible linker that connects the fold with theC-terminal tRNA binding domain.13 Importantly,none of the residues involved in the cofactor-binding or catalysis in TrmD are in common withthose identified in MJ0883, suggesting that TrmDand MJ0883/Trm5 are unrelated not only insequence but in structure.

Implications for evolution

AdoMet is one of the most ancient and widelyused enzyme substrates and is even used byRNAs in riboswitches to regulate gene expres-sion.42 The AdoMet-dependent protein MTases areinvolved in wide ranging activities, includingbiosynthesis, signal transduction, protein repair,chromatin regulation, and gene silencing.23 TheseMTases have used as many as five differentAdoMet folds to catalyze their reactions. Whilemany MTases use the same AdoMet folds tocatalyze diverse reactions, most commonly theclass I fold, the examples of the same activitycatalyzed by different AdoMet folds are farless common. Here, the MJ0883-like archaealtRNA(m1G37) MTase and eukaryotic Trm5 areshown to use the class I AdoMet fold, whereas thebacterial TrmD uses the class IV fold. This is anexample of inventing two distinct folds to catalyzethe same reaction. The development from the twodistinct folds to the separate tRNA(m1G37)MTases, MJ0883/Trm5 and TrmD, appears inde-pendent for the two classes. For example, analysisof a BLAST search has indicated that the MJ0883/Trm5 group contains the tRNA-binding domain atthe N-terminal side of the AdoMet fold, and aguanine-binding site at the C-terminal side. Thesetwo domains were identified based on similaritiesto respective domains in the Asp/Glu-tRNAamidotransferases and the guanine nucleotide-binding proteins. In contrast, TrmD containsthe tRNA domain at the C-terminal side of theAdoMet fold. The invention of two distinctenzymes to catalyze the tRNA(m1G37) modifi-cation was previously unexpected, given that themodification is conserved in bacteria, eukarya,and archaea. It is also of evolutionary interest,because most of the RNA modification enzymesare conserved in all three life forms.

The separation of MJ0883/Trm5 from TrmD is aremarkable example of non-orthologous gene dis-placement, where an important enzyme functionis performed by two proteins of differentsequences.43 This is in contrast to the two struc-turally distinct classes of aminoacyl-tRNA synthe-tases that catalyze aminoacylation of tRNA,44,45


where synthetases of the same specificity are ortho-logous and are descended from a common ancestorof a specific class. For example, the synthetases forisoleucine are universally class I,46 whereas thosefor alanine are universally class II,47 regardless ofthe taxonomic origins. The only exception is thesynthetases for lysine, which exist as a class I inarchaea and some bacteria, but as a class II ineukarya and most bacteria.48,49 In the case of thetRNA(m1G37) MTases, however, the phyleticpatterns of the non-orthologous class I and classIV folds show striking complementarities: theclass I-specific MJ0883/Trm5 is present in allarchaea and eukarya, whereas the class IV-specificTrmD is present in all bacteria. This demonstratesclearly both the principle of non-orthologous genedisplacement and the ancient split of archaea–eukarya from bacteria.

This study has offered new promises for drugdesign to target the important tRNA(m1G37)MTase in pathogenic bacteria. The sequence andstructural distinction of bacterial TrmD from theeukaryotic Trm5 suggests that strategies can bedeveloped to specifically inhibit TrmD without adeleterious effect on humans. The AdoMet-bindingsite is an attractive target for small molecular com-pounds, where the intricacy of the unique trefoilknot in TrmD can be fully exploited to developdifferent properties of ligands. Also, the catalyticsite of TrmD is so different from that of Trm5 suchthat it may require a different mechanism forrecognition of the tRNA G37 base for methylation.In addition, TrmD must function as a dimer,whereas the eukaryotic Trm5/MJ0883 can functionas a monomer (Figure 5(A)). The dimer interfacein crystal structures of TrmD is expansive and isintegral to the enzyme function, thus offeringanother well-endowed target for inhibition.Further studies of MJ0883/Trm5 in comparisonwith TrmD will undoubtedly yield additionalinsights for drug discovery.

Materials and Methods

Enzyme purification, methylation assay,and complementation

Mj0883 was cloned by PCR and the sequence of thegene was confirmed by sequence analysis. The gene wasthen introduced into the expression vector pET-22b inthe Nde I and Bam H1 sites and the protein was expressedin E. coli BL21(DE3) with a C-terminal His-tag and waspurified through a heat-step, a metal-affinity column(Talon, ClonTech), and monoS on an FPLC. MJ0883(1 mM) was assayed at 50 8C in 0.1 M Tris–HCl (pH 8.0),4 mM DTT, 0.1 mM EDTA, 6 mM MgCl2, 0.1 M KCl,0.024 mg/ml bovine serum albumen (BSA), using50 mM [3H]AdoMet (0.02 mCi/ml, 870 dpm/pmol) as thecofactor and 0.5–10 mM M. jannaschii tRNACys transcriptas the substrate. Aliquots of the reaction were removedat various time points, precipitated by 5% (w/v) tri-chloroacetic acid (TCA) on filter pads, washed withethanol and ether, and air-dried before scintillationcounting. Steady-state kinetic parameters for AdoMet

were obtained by ranging the cofactor at 0.5–20 mM,while fixing the tRNA at 6 mM, and those for tRNAwere obtained by ranging the substrate at 0.5–10 mM,while fixing the cofactor at 50 mM. Site-directedmutations in MJ0883 were created by the QuikChangeprotocol and were confirmed by sequence analysis.Mutant enzymes were purified by the same procedureas the wild-type and were assayed by their initial ratesof methyl transfer under steady-state conditions, wherethe tRNACys transcript was at 1 mM, AdoMet at 50 mM,and mutant enzymes at 3 mM. To test complementationof the ts mutant of S. typhimurium GT5454, the wild-typegene Mj0883 was isolated from pET-22b with Xba Iand HindIII, the sticky Xba I end was filled in by Klenow,subcloned into the Sma I and HindIII sites of pKK223-3,and tested for growth complementation in GT5454. Eachstrain was streaked on an LB plate containing ampicillin(100 mg/ml) and IPTG (1 mM) and incubated at 30 8C or42 8C for 16 hours. Cell lysate of GT5454 was preparedfrom strains grown at 30 8C to the stationary phase,sonicated in the assay buffer, cleared of debris bycentrifugation, and dialyzed before assay for activity.

Analysis of methylated tRNA

For the nuclease P1 digestion, radiolabeled tRNA tran-scripts were obtained by in vitro transcription, using[a-32P]GTP, and methylated by MJ0883, followed by PI

digestion (0.03 unit/ml) at 50 8C for one hour in 20 mMsodium citrate (pH 5.3), 0.1 M ZnCl2. An aliquot wasapplied to a thin layer cellulose plate (Fisher; 5716-7)and chromatographed in the first dimension (isobutyricacid/ammonia/water, 66 : 1: 33, by volume) and thenthe second dimension (0.1 M phosphate buffer (pH 6.8),with 2% (v/v) n-propanol, and 60% (w/v) ammoniumsulfate). Labeled nucleotides were localized by auto-radiography and quantified by a PhosphorImager. Forthe nuclease T1 digestion, tRNA was labeled at the 30

end by the E. coli CCA-adding enzyme, using[a-32P]ATP, methylated by MJ0883, and digested by T1

(Roche; 109193) in 20 mM sodium citrate (pH 5.5), 1 mMEDTA, at 50 8C for ten minutes. In primer extension,the primer was complementary to C43 to A57 ofM. jannaschii tRNACys, labeled at the 50 end, annealed tothe tRNA and extended by AMV reverse transcriptase(Roche; 1495062) in 50 mM Tris–HCl (pH 8.5), 8 mMMgCl2, 30 mM KCl, 1 mM DTT, 40 mM potassium borateat 42 8C for 30 minutes. Extension was quenched withEDTA, loaded onto a Sepak column (Waters; 054955)that has been washed with acetonitrile, acetonitrile/water, and water. Extension products were elutedby acetonitrile/methanol/water (7 : 7 : 6, by volume),dried, and separated on denaturing PAGE.

Biochemical analysis

The gel shift assay of tRNA binding was in the bind-ing buffer of 25 mM sodium phosphate (pH 6.8), 75 mMKCl, 10 mM MgCl2, as described.50 The fraction ofbound and free tRNA in each binding reaction wasanalyzed by PhosphorImager analysis, using thesoftware ImageQuant (Molecular Probes). The tRNAbinding data were fitted by solving the mass conser-vation equations and by estimating Kd values simul-taneously, using the IGOR software (WaveMetrics, Inc.).Data set that was used to fit were (1) E-tRNA versus Etotal;(2) E2-tRNA versus Etotal; and (3) tRNAbound ( ¼ E-tRNA þE2-tRNA) versus Etotal. The mass conservation equations


were used as follows:

½tRNAtotal ¼ ½tRNAfree þ ½E-tRNA þ ½E2-tRNA ð1Þ

½Etotal ¼ ½Efree þ ½E-tRNA þ 2½E2-tRNA ð2Þ

Kd1 ¼ ½tRNA½E=½E-tRNA ð3Þ

Kd2 ¼ ½E-tRNA½E=½E2-tRNA ð4Þ

By combining equations (1)–(4):

½tRNAtotal ¼ ½tRNAfree þ ½E½tRNA=Kd1

þ ½E2½tRNA=Kd1=Kd2 ð5Þ

½Etotal ¼ ½Efree þ ½E-tRNA þ 2½E2½tRNA=Kd1=Kd2 ð6Þ

Kd1 and Kd2 were estimated by simultaneously fitting thedata sets (see below) to equations (5) and (6) using theIGOR software.

Gel filtration of MJ0883 on the Suprose 12 sizingcolumn was in the same buffer as that of gel shift. Themolecular mass markers (BioRad) used were thyro-globulin (670 kDa), bovine g-globulin (158 kDa), chickenovalbumin (44 kDa), equine myoglobin (17 kDa), andvitamin B12 (1.4 kDa).

Bioinformatics and phylogenetic analysis

MJ0883 and the S. cerevisiae Trm5 were used as thequery sequences to identify, respectively, the archaeaand eukaryotic homologs by the BLAST and PSI-BLASTsearches of the SwissProt and unfinished genomedatabases. Eight archaea homologs of Mj0883 wereidentified and their accession numbers (in parentheses)were: Mj1557, Methanobacterium thermoautotrophicum,Mth(O27268); Methanosarcina mazei, Mz(Q8PW27);M. acetivorans, (Ma) Methanopyrus kanderi (Q8TWU2);H. halobium, Hh(Q9HMM2 and Q9HQK5); Archaeoglobusfulgidus (O28306); Pyrococcus furiosus, (Pf)(Q8U4J6).Several eukaryotic candidates were identified:Neurospora crassa, (Nc)(JC4255); G. intestinalis; A. thalania(At); D. melanogaster (Dm); Anopheles gambiae,(Ag)(EAA10891); Plasmodium falciparum (NP704683);Encephalitozon cuniculi (NP584745); Leishmania major(AAM68974); Plasmodium yoellii yoellii (EAA15979). Toestablish the relationships of MJ0883/Trm5 with theclass I MJ0882, the entire protein sequences were alignedby CLUSTALW1.7,51 using PAM matrices with manualadjustments. A phylogenetic tree was constructed bythe neighbor-joining method using ClustalW for align-ment and Treeview (v.1.6.6) for visualization, andwas confirmed by separately constructing a maximumparsimony tree using PHYLIP’s Protpars program.

To determine the AdoMet fold of MJ0883, the entireprotein sequence was aligned to the five known classesof MTases. The class I enzymes used in the alignmentwere Hha I, Hae III, Taq I, Pvu II, FtsJ, and DNMT2. Theclass II enzymes were Met H of E. coli, S. typhimurium,H. influenzae, M. tuberculosis, H. sapiens, C. elegans, andR. novartis. The class III enzymes were the cobalt-per-corrin transmethylase (CbiF). The class IV enzymes wereTrmD of E. coli, H. influenzae, S. typhimurium, B. subtilis,B. borgdorferi, B. halodurans, A. aerolicus, P. aeruginosa,T. pallidium, and RlmB of E. coli. The class V enzymeswere the histone H3 MTase and ribulose-1,5 biphosphatecarboxylase/oxygenase large subunit N-MTase. Thealignment was used to generate a phylogeny using theneighbor-joining program and bootstrap analysis in

CLUSTALW and maximum parsimony using Protparsfrom PHYLIP.

Acknowledgements

We thank Dr Glenn Bjork for the strain GT5454and pUMV4 plasmid; Dr W. Michael Holmes forthe clone of E. coli TrmD and tRNALeu; Dr BarryCooperman and Hyuk-Soo Seo for curve-fit of gelshift analysis; Dr Xiaodong Cheng for commentson the manuscript; and Dr Jim R. Brown andDr. Eugene Koonin for discussion of sequencealignment. Supported by NIH grant (GM066267 toY.M.H.).

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Edited by D. E. Draper

(Received 18 February 2004; received in revised form 4 April 2004; accepted 5 April 2004)


Distinct Origins of tRNA(m1G37) Methyltransferase

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