INTRODUCTION
Transfer RNAs (tRNAs) are fundamental for translation of the genetic code. Although these nucleic acids are transcribed with only four nucleotides (A, C, G, U), the diversity of the four building blocks is substantially expanded by post-transcriptional modifications, which enhance the structure and activity of the L-shaped molecules in all living cells ( 1 ). Collectively, more than 100 different chemical moieties have been introduced to modify tRNAs in all three domains of life ( 2 ). Each chemical moiety is created after transcription by an enzyme or a pathway of enzymes. Most of these modifying enzymes target position 34 of tRNA, the wobble position of the anticodon, or position 37, on the 3′-side of the anticodon, to produce chemical moieties that improve the quality of decoding. Although the cellular genomic space dedicated to genes for tRNA modification enzymes is large, few of these enzymes are understood at the mechanistic level. A critical barrier to progress is the lack of the ability to isolate tRNA molecules to study modifications, particularly when a tRNA is required for growth and cannot be readily changed with mutations for enzymatic analysis. Another critical barrier is the insufficient understanding of the inter-dependence between positions 34 and 37 when each harbors a distinct modification. Little is known whether the two modifications are independent of each other or whether one determines the activity of the other. Overcoming these critical barriers is an important step forward to address the biology of tRNA in living cells.
We focus on the cmo 5 modification of the U34 wobble base in Gram-negative tRNAs ( 3 ), which is associated with isoacceptors specific for six amino acids: Ala, Ser, Pro, Thr, Leu and Val ( 4 , 5 ). While the unmodified U34 can read all four nucleotides in bacteria ( 6–10 ), the cmo 5 modification improves the quality of reading each ( 11–15 ) while the additional s 2 modification restricts the reading specificity to A, G and less so U ( 16 ). The cmo 5 U modification is synthesized via multiple enzymatic reactions (Figure 1A ). First, U34 is hydroxylated at the 5-position to form 5-hydroxyU (ho 5 U34) by an as-yet unknown enzyme ( 15 ). Second, ho 5 U34 is converted to cmo 5 U34 by the combined action of two S -adenosyl methionine (AdoMet)-dependent enzymes ( 17 ). Specifically, CmoA transfers the carbon dioxide of prephenate to the sulfonyl methyl of AdoMet to generate carboxy- S- AdoMet (Cx-AdoMet), and subsequently CmoB transfers the carboxy-methyl of Cx-AdoMet to the 5-hydroxy of ho 5 U34 to synthesize cmo 5 U34. Because prephenate is derived from chorismate, biosynthesis of cmo 5 U is linked through chorismate to biosynthesis of aromatic amino acids and vitamins ( 18 , 19 ). Interestingly, the carboxyl of cmo 5 U34 in four of the six families of tRNA isoacceptors (Ala, Ser, Pro, Thr) is further methylated to mcmo 5 U34 (Figure 1A ) in a terminal methyl transfer reaction catalyzed by CmoM ( 5 ). The presence of the methyl ester in mcmo 5 U34 further ensures reading frame accuracy by suppressing +1-frameshifts ( 5 ). However, the selectivity for four out of six families of tRNAs by CmoM is not understood at the molecular level, thus limiting our insight into the cmo 5 U34 pathway.
The four tRNA families with the terminal methyl group in mcmo 5 U34 share G35 in the middle position of the anticodon, whereas the two without the methyl group share A35 in common ( Supplementary Figure S1 ). This raises the hypothesis that G35 is a major determinant for recognition by CmoM. In the crystal structure of the ribosomal 30S subunit in complex with an A-site-bound anticodon–stem–loop of tRNA Val , which lacks the terminal methylation, the carboxylate of the unmethylated cmo 5 U34 in tRNA Val interacts with the N 6 of the neighboring base A35 to stabilize the wobble pairing with all four bases at the third position of the codon ( 14 ). If A35 is replaced with G35, the N 6 is replaced with an O 6 , which would block the interaction. The addition of a methyl ester to the cmo 5 moiety in the form of mcmo 5 U34 would neutralize the repulsion between the O 6 and the negative charge of the carboxylate, thus providing a basis for the hypothesis that the terminal methylation is necessary for stabilizing the pairing with anticodons that contain G35. However, testing and validating the hypothesis has not been done and the difficulty is associated with at least two reasons. First, the substrate for CmoM, cmo 5 U34-tRNA, must be isolated from cells in a cmoM -knockout ( cmoM-KO ) background. An in vitro reconstitution of the cmo 5 U34 substrate from a tRNA transcript is not yet possible due to the lack of information on the enzyme that synthesizes ho 5 U34. Second, tRNA isoacceptors harboring cmo 5 U34 are usually essential for cell growth (e.g. those for Ser, Leu, Pro and Thr) and cannot be readily made with a site-specific substitution inside of cells without compromising cell survival.
We show here the development of a genetic approach ( Supplementary Figure S2 ) that enables isolation of mutants of Escherichia coli tRNA Pro and tRNA Val with a site-specific substitution at position 35 of the anticodon. This approach is robust and amendable to generate sufficient quantities of tRNA for detailed biochemical and mechanistic studies. It is broadly applicable to isolate mutants of other tRNA species that are essential for growth. Using this approach, we show that substitution of G35 in the UGG isoacceptor of tRNA Pro (UGG tRNA Pro ) abolishes the terminal methyl transfer by CmoM, while introduction of G35 to the UAC isoacceptor of tRNA Val (UAC tRNA Val ) confers the activity, supporting the notion that G35 is a major determinant for tRNA recognition by CmoM. Additionally, using tRNA Pro isolated from this approach, we explore the inter-dependence of the primary m 1 G methylation at position 37 present in this tRNA ( 20 ) and the terminal methylation at position 34. We show that the m 1 G37 methylation is important for the terminal methylation at position 34 but not vice versa , providing new insight into the ranking and ordering of the two modifications in the cellular production of a functional tRNA.
MATERIALS AND METHODS
tRNA plasmids
All primers for creating tRNA plasmids are listed ( Supplementary Table S1 ). The maintenance plasmid for expression of a chimeric tRNA was based on pACYC184. The gene for the G34U mutant of the isoacceptor GGG tRNA Pro (G34U- proL , Supplementary Figure S3 ) with the lpp constitutive promoter and the rrnC transcription terminator sequence was isolated from a derivative of pGFIB ( 21 , 22 ) as a PvuII fragment and cloned into the EcoRV site of pACYC. The G34U mutation was created with Quikchange (Agilent) using primers GGGtoUGG-F and GGGtoUGG-R. The gene for the G34U mutant of the isoacceptor GAC tRNA Val (G34U- valW , Supplementary Figure S4 ) was created by hybridization of a pair of oligos GACtoUAC-F and GACtoUAC-R, inserted into the EcoRI and PstI sites of pGFIB, isolated as a PvuII fragment with the lpp promoter and rrnC terminator sequence, and cloned into the EcoRV site of pACYC184. The Bacillus subtilis tRNA Cys/GCA ( BscysT ) was amplified from pTFMA ( 23 ) using primers BscysT-F and BsCysT-R and cloned into pGFIB at EcoRI and PstI sites. The PvuII fragment encoding the gene, the lpp promoter, and the rrnC terminator, was sub-cloned to pACYC184 at the EcoRV site to generate a cysT-KO maintenance plasmid.
The test plasmid for expression of a wild-type (WT) or a mutant tRNA was based on pKK223–3. The gene for UGG tRNA Pro (encoded by proM ) or UAC tRNA Val (encoded by identical genes valUXY and valTZ ) was cloned into the EcoRI and PstI sites. Anticodon mutants were created by Quikchange with primers UGGtoUAG-F and UGGtoUAG-R for tRNA Pro and primers UACtoUGC-F and UACtoUGC-R for tRNA Val . The amber suppressor mutant of E. coli cysT on pGFIB was made by Quikchange using primers GCAtoCUA-F and GCAtoCUA-R, which changed the anticodon from GCA to CUA.
E. coli strains
All of the E. coli strains in this study are listed ( Supplementary Table S2 ). The cmoB-KO locus of E. coli cmoB-KO strain (from Dr. Steven Almo) was transferred to the published E. coli trmD-KO strain maintained by trm5 ( 24 ). After transfer, the kanamycin marker ( kan R ) of the cmoB-KO locus was removed by the flippase recombinase (FLP) encoded in pCP20 ( 25 ). To construct the vehicle for expression of a mutant version of UGG tRNA Pro ( Supplementary Figure S3 ), E. coli JM109 was used as the host and transduced with the P1 lysate of an E. coli cmoM-KO strain (Coli Genetic Stock Center, Yale University) and removed of the kan R of the cmoM-KO locus by FLP. The resulting JM109- cmoM-KO strain was introduced with the maintenance plasmid expressing the chimeric G34U- proL . The strain was then deleted of the UGG tRNA Pro gene, using the λ Red recombinase of pKD46 ( 26 ) to catalyze homologous recombination at the proM locus with a proM-kan R PCR fragment amplified by primers proMKO-F and proMKO-R from pKD4. The kan R for selection of the resultant JM109- cmoM-KO-proM-KO strain was removed by FLP. The resultant strain was transformed with the test pKK223–3 plasmid with proM to generate the expression vehicle for a mutant version of proM .
To construct the expression vehicle of a mutant form of UAC tRNA Val ( Supplementary Figure S4 ), the E. coli JM109- cmoM-KO strain above was introduced with the maintenance plasmid carrying the chimeric G34U- valW. E. coli JM109- cmoM-KO carries five genes for tRNA Val ( valUXY and valTZ , each encoding the same sequence, and was made into two derivatives, one deleted of the cluster of valUXY (using primers valUXYKO-F and valUXYKO-R), and the other deleted of the cluster of valTZ (using primers valTZKO-F and valTZKO-R). The kan R of the first resultant strain JM109- cmoM-KO-valUXY-KO was removed by FLP and the strain was transduced with the P1 lysate of the second resultant strain JM109- cmoM-KO-valTZ-KO to generate the JM109- cmoM-KO-valUXYTZ-KO strain. After removal of kan R by FLP, the resultant strain was transformed with the test pKK223–3 plasmid carrying the gene for UAC tRNA Val as a vehicle for expressing a mutant version of the tRNA. To construct the E. coli tRNA Cys/GCA ( cysT )- KO ( Supplementary Figure S5 ), the kan R (amplified by PCR from pKD4) was used to target the cysT chromosomal locus in JM109 using primers cysTKO-F and cysTKO-R. After the resultant strain was introduced with a maintenance plasmid expressing BscysT , the cysT was removed by λ Red recombinase and the kan R removed by FLP. This cysT-KO locus , together with the maintenance plasmid for expressing BscysT and a pGFIB-derived test plasmid for expressing the amber-reading form of cysT , was transduced into an E. coli XAC-1 strain with an internal amber codon in the chromosomal lacZ ( 21 ).
Growth assay
Strain JM109- cmoM-KO-proM-KO maintained by chimeric G34U- proL , strain JM109- cmoM-KO-valUXYTZ-KO maintained by chimeric G34U- valW , and strain JM109- cmoM-KO-cysT-KO maintained by BscysT , were cultured in LB medium overnight at 37°C. Cells were then inoculated into 25 ml LB at 1:100 at 37°C and the OD 600 was monitored over time using Tecan Infinite M200 Pro plate reader (Tecan). The growth assay for strain JM109- cmoM-KO-proM-KO maintained by chimeric G34U- proL , harboring either pKK223–3-G35A- proM or an empty pKK223–3, was performed similarly except that the overnight culture was inoculated to 30 ml fresh LB to OD = 0.04 and 0.4 mM IPTG was added at T = 2 h. Overnight cultures of the two strains were also serially diluted and spotted on LB plates with Amp and Chl and 0.4 mM IPTG and incubated at 37°C.
X-gal assay
The E. coli strain XAC-1 cysT-KO maintained by pACYC184- BscysT was transformed with the pGFIB- cysT plasmid either with GCA or CUA anticodon, and streaked and grown on an M9 plate containing 20 μg/ml 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal) at 37°C overnight. The amber suppression activity was monitored as the color development by the β-galactosidase-catalyzed hydrolysis of X-gal.
Recombinant proteins
The E. coli cmoM gene was amplified from E. coli MG1655 genomic DNA using primers cmoM-F and cmoM-R and cloned into pET28 (Novagen) at NdeI and NotI sites. This pET28- cmoM was transformed into E. coli BL21(DE3) and over-expressed for 4 h at 37°C in LB containing 0.1 mM IPTG. The dual 6xHis-tagged recombinant CmoM was purified with HisLink Protein Purification Resin (Promega) as described previously ( 5 ). The recombinant E. coli TrmD was prepared as described ( 27–29 ). The recombinant E. coli prolyl-tRNA synthetase (ProRS) and leucyl-tRNA synthetase (LeuRS) were purified as described ( 30 ).
Substrate tRNAs
All substrate tRNAs for this study were expressed from the tac -controlled pKK223–3 plasmid and isolated from E. coli cells. Each tRNA was pulled down from total RNA by an affinity resin coupled with an oligonucleotide complementary to a sequence of the target tRNA ( 24 , 31 , 32 ). For tRNA Val , due to its partial sequence homology with UGC tRNA Ala , two successive affinity purification steps were performed. The first used the affinity oligo Val-AP1 and the second used the affinity oligo Val-AP2 ( Supplementary Table S1 ). The quality of UGG and UAG tRNA Pro in cmo 5 U34- or mcmo 5 U34-modified state is shown as a single band in denaturing gel analysis ( Supplementary Figure S6A ), indicating homogeneity.
WT tRNA Pro and tRNA Val were each purified from the over-expression plasmid pKK223–3 driven by the tac promoter in cmoM-KO cells. To isolate UGG tRNA Pro lacking m 1 G37 but containing a distinct state of the wobble modification, we used E. coli strain MG1655 trmD-KO as the basis ( 24 ). The trmD-KO strain was made into cmoB-KO or cmoM-KO by P1 transduction and the kan R was removed. Cells were then transformed with pKK223–3- proM and grown to saturation in an overnight culture with 0.2% (w/v) arabinose (Ara) to allow expression of trm5 . Cells were then freshly diluted into Ara-free LB at 1:100 and grown for 3 h, followed by a second dilution into fresh Ara-free LB at 1:5 and growth for 2 h. After these two dilutions, cells were harvested and primer extension analysis showed that the cellular level of m 1 G37-tRNA was below 5%.
Kinetic analysis
Methylation or aminoacylation was assayed at 37°C by monitoring the incorporation of [ 3 H-methyl] of AdoMet or [ 3 H]-amino acid to tRNA as acid precipitable counts ( 33–37 ). Aliquots were removed over time and acid-precipitable counts measured. Data for steady-state assays of V o as a function of tRNA concentration were fit to the hyperbolic Michaelis–Menten equation [ y = m 1 × x /( m 2 + x )] ( 38 ), where m 1 and m 2 were k cat and K m , respectively. Data for single-turnover assays of a time course were fit to a single exponential equation [ y = m 1 × (1 – exp(– m 2 × x ))] ( 38 , 39 ), where m 1 and m 2 were the plateau level and the k obs , respectively. Data of k obs as a function of enzyme concentration were fit to the hyperbolic Michaelis–Menten equation.
For CmoM methylation, steady-state assays of plateau levels were performed with 1.0 μM enzyme, 1.0 μM tRNA, and 20 μM [ 3 H-methyl]-AdoMet in a buffer of 50 mM HEPES–KOH (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , and 7 mM β-mercaptoethanol. Substrate tRNAs were isolated from a vehicle strain expressing E. coli WT or a mutant version of UGG tRNA Pro or UAC tRNA Val . Single-turnover assays were performed with 1–16 μM enzyme, 1.0 μM tRNA, and 20 μM [ 3 H-methyl]-AdoMet in the same buffer. The substrate UGG tRNA Pro with m 1 G37 was isolated from a vehicle strain expressing the WT tRNA, whereas the substrate UGG tRNA Pro without m 1 G37 was isolated from the same vehicle strain with trmD-KO maintained by the Ara-controlled trm5 on pACYC, which also expressed the lpp -controlled chimeric G34U mutant of GGG tRNA Pro .
For TrmD methylation, steady-state assays were performed with 10 nM enzyme, 0.5–12.5 μM tRNA, and 28.9 μM [ 3 H-methyl]-AdoMet in a buffer of 100 mM Tris–HCl, pH 8.0, 24 mM NH 4 Cl 2 , 4 mM DTT, 0.024 mg/ml BSA, and 6 mM MgCl 2 . The three states of UGG tRNA Pro were isolated from an E. coli trmD-KO strain ( 24 ) that expressed the tRNA from the pKK223–3 plasmid. The ho 5 U34-state was isolated from the strain with additional cmoB-KO (with the kan R removed), the cmo 5 U34-state was isolated from the strain with additional cmoM-KO , and the mcmo 5 U34-state was isolated from the trmD-KO strain.
For aminoacylation with Pro, the UAG variant of tRNA Pro/UGG was isolated from E. coli Pro vehicle strain ( Supplementary Table S2 ). Plateau charging assays were conducted using 1.0 μM ProRS, 1.0 μM tRNA Pro/UAG , 20 μM [ 3 H]-Pro in a buffer of 20 mM KCl, 50 mM HEPES pH 7.5, 4 mM DTT, 0.2 mg/ml BSA, 10 mM MgCl 2 , and 2 mM ATP. For aminoacylation of the tRNA with Leu, LeuRS and [ 3 H]-Leu were used instead.
LC–MS/MS analyses
WT and mutant tRNA Pro species (3 μg), purified from cells, were digested with RNase T1 (50 U/μg tRNA) in a 220 mM ammonium acetate buffer at 37°C for 2 h. Upon completion, the samples were lyophilized and resuspended in 10 μl of the mobile phase A buffer (8 mM TEA/220 mM HFIP, pH 7.0). For liquid chromatography and mass spectrometry (LC–MS/MS) analysis, tRNA fragments from T1 digestion were separated in a Poroshell 120-EC-C18 column (1 × 50 mm, 2.7 μm particle size) using mobile phase A and mobile phase B (50% buffer A in methanol). The column oven was set at 60°C with the Thermo Surveyor HPLC attached to the Thermo LTQ-XL linear ion trap mass spectrometer. The LC gradient starts with 5–95% mobile phase B in 45 min at a flow rate of 60 μl/min. Post column equilibration is set for 10 min prior to the next injection. The MS operating parameters are: capillary temperature of 275°C, spray voltage of 3 kV and sheath, and auxiliary and sweep gases at 35, 14, 10 arbitrary units respectively. Data were recorded in negative mode, with a scan range from 600 to 2000 m/z . The product ion scans were obtained using collision induced dissociation at the normalized collision energy of 35%. All of the predicted tRNA sequences were obtained from the genomic tRNA database ( http://gtrnadb.ucsc.edu/ ). The prediction of m/z and fragmentation of each was calculated by the Mongo Oligo Mass Calculator ( http://mods.rna.albany.edu/masspec/Mongo-Oligo ).
RESULTS AND DISCUSSION
A genetic approach
We chose E. coli UGG tRNA Pro , encoded by the proM gene, as an example. This isoacceptor is a substrate for CmoM to convert cmo 5 U34 to mcmo 5 U34 (Figure 1B ) ( 5 ). The other Pro isoacceptors in E. coli contain the GGG and CGG anticodon, respectively. Of the three, the UGG isoacceptor is essential for growth ( 15 ), the elimination of which would leave cells unable to efficiently translate the Pro codon CCA, ultimately leading to cell death. This growth-essentiality makes it impossible to introduce mutations to G35 to test recognition by CmoM. To overcome this limitation, we developed a genetic approach ( Supplementary Figure S2 ), in which we created a plasmid-borne chimeric tRNA Pro to maintain cell growth, enabling us to remove the native proM gene from the chromosome while expressing a test version of the proM gene from a second plasmid for isolation of its tRNA product for enzymatic analysis. This approach consists of three key components. First, a chimeric tRNA Pro was expressed from the strong and constitutive lpp promoter in a pACYC-derived maintenance plasmid ( 21 ). The chimera was based on the sequence of the GGG isoacceptor (encoded by the proL gene, Figure 1C ) but was made with the G34U mutation to carry the UGG anticodon for reading the Pro codon CCA. The remaining Pro codons would be read by the other two isoacceptors whose genes were intact on the chromosome. Second, the native proM was removed from its chromosomal locus in an operon containing tRNA genes argX, hisR , and leuT ( Supplementary Figure S3 ). This was achieved in an E. coli cmoM-KO strain by λ Red recombination ( 26 ). The cmoM-KO strain was necessary as the host to produce tRNA species containing the cmo 5 U34 wobble base but lacking the terminal methylation for evaluation as a substrate for CmoM. Third, the test version of proM was then expressed from pKK223–3 that was compatible with the maintenance pACYC. Expression of the test proM was driven from the IPTG-inducible tac promoter to produce high levels of the tRNA product for purification and kinetic analysis. In this 3-component system, expression of the maintenance chimeric tRNA Pro in proM-KO cells was sufficient to promote growth (Figure 1D ), although the growth was slower relative to cells with proM intact on the chromosome. This establishes an E. coli vehicle strain that could be used to produce a mutant version of the proM tRNA from the test plasmid without losing cell viability.
Using a similar genetic approach, we created an E. coli vehicle strain that was used to produce a mutant version of the UAC tRNA Val ( Supplementary Figure S4 ). The WT isoacceptor is encoded in five genes of the same sequence on the chromosome ( valU, valX, valY, valT and valZ ), each of which produces a tRNA transcript that is modified to cmo 5 U34 without the CmoM-catalyzed terminal methylation ( 5 ). In this vehicle strain, all of the isoacceptor genes were removed from the chromosome, while cell viability was maintained by expression of a chimeric tRNA Val based on the sequence of the valW -encoded GAC isoacceptor but made to contain the UAC anticodon. Growth of cells maintained by the chimeric tRNA Val was only slightly slower relative to cells containing valUXYTZ genes (Figure 1D ). Expression from the test plasmid that harbored a mutant form of one of the valUXYTZ genes then provided a mechanism to isolate the mutant tRNA for enzymatic analysis.
We showed that this genetic approach was applicable to other single-gene tRNA species. For example, cysT is a single gene for GCA tRNA Cys in E. coli . Using the same genetic approach, we created a cysT-KO vehicle strain that was maintained viable by the homologous gene of BscysT in a maintenance plasmid and able to express the amber-reading version of cysT from a test plasmid ( Supplementary Figure S5 ). The viability was consistent with our finding that BscysT tRNA is readily charged by the endogenous E. coli cysteinyl-tRNA synthetase (CysRS) ( 23 ). The amber-suppressor mutant of E. coli cysT was used to test its adaptor activity during live-cell protein synthesis. Indeed, the cysT-KO vehicle system, when introduced to strain XAC-1 harboring lacZ with an amber mutation, conferred a blue color on X-gal indicator plates ( Supplementary Figure S5 ), indicating suppression of the amber mutation. These data demonstrate the adaptor activity of the amber-reading test tRNA Cys during protein synthesis in an E. coli strain where the essential single-gene cysT has been eliminated, validating the broad utility of our genetic approach.
G35 as the major determinant for recognition by CmoM
With the genetic approach in hand, we tested the importance of G35 as a determinant for tRNA recognition by CmoM. We created an E. coli vehicle strain that produced the G35A mutant of UGG tRNA Pro and a separate strain that produced the reciprocal A35G mutant of UAC tRNA Val (Figure 2A and B ). Each mutant tRNA, as well as its WT counterpart (over-expressed from the pKK223–3 plasmid), was expressed in the cmoM-KO strain to abort the terminal methylation and to accumulate tRNA in the cmo 5 U34 precursor state. Each was produced to high levels and purified to homogeneity ( Supplementary Figure S6A ) from binding to a complementary oligonucleotide on a solid support. As shown by LC-MS/MS, each purified tRNA contained cmo 5 U34 in the precursor state, as exemplified by the UAG variant of tRNA Pro (Figure 2C , Supplementary Figure S6B ), and was used as a substrate for CmoM.
Recombinant E. coli CmoM was over-expressed with a His-tag at both the N- and C-termini and purified to greater than 95% homogeneity. Using [ 3 H-methyl]-AdoMet as the methyl donor, we monitored the CmoM-catalyzed incorporation of [ 3 H-methyl] from the donor to tRNA as acid-precipitable counts on filer pads as we have shown with other AdoMet-dependent methyl transferases ( 27–29 , 38–40 ). Assays in a steady-state multiple-turnover condition showed that the G35 base in the anticodon is indeed the major determinant for recognition and discrimination of tRNA by CmoM. While the WT UGG tRNA Pro was fully methylated by CmoM, the single G35A substitution abolished the methylation (Figure 2D ). Conversely, while the WT UAC tRNA Val was an inactive substrate for CmoM, the single A35G substitution was sufficient to confer full methylation to this tRNA (Figure 2E ). Thus, despite in different sequence contexts of tRNA Pro and tRNA Val , the G35 base alone determines the methylation by CmoM.
To more closely evaluate the role of G35 in the methylation by CmoM, we determined the kinetic parameters of individual tRNA species (Figure 2F , G ). CmoM is a member of the class I AdoMet-dependent methyl transferases (PDB: 4HTF), which use the Rossmann dinucleotide-fold to accommodate the methyl donor ( 41 , 42 ). We had shown that enzymes of the Rossmann-fold are rate-limited by the release of products ( 30 , 38 , 43 ), rather than by the chemistry involving the S N 2 nucleophilic attack on the sulfonium center of AdoMet. This kinetic signature indicates that the rate of chemistry is faster relative to the release of products and that, to probe the chemistry, kinetic assays must be performed in single-turnover assays to monitor chemistry, rather than in steady-state assays to monitor product release ( 38 ). We developed single-turnover assays for CmoM, where the enzyme was in excess of each tRNA to permit rapid equilibrium binding in one turnover, such that the rate constant k obs of the turnover was not limited by binding but by the chemistry of methyl transfer ( 44 ). A titration of k obs as a function of enzyme concentration then provided the basis to determine the saturating k obs as k chem (the intrinsic rate constant of methyl transfer) and the enzyme concentration that achieved half of the saturation kinetics as the thermodynamic dissociation constant K d for the tRNA.
Analysis of k obs as a function of CmoM concentration for the WT UGG tRNA Pro showed that all data points of single-turnover assays were fit to a hyperbolic equation without a lag phase (Figure 2F ), supporting the notion that methyl transfer was performed in rapid binding equilibrium. The k obs of each turnover was a composite term, representing all of the steps from binding to methyl transfer, including a conformational transition of the enzyme-tRNA-AdoMet complex that might have occurred before chemistry. The absence of a lag phase indicated that all steps leading to and including the chemistry were fast and that the kinetics was limited by the rate of methyl transfer. While we were unable to determine the kinetics of the UAG mutant of tRNA Pro or the WT UAC tRNA Val , due to extremely low levels of activity, we were able to measure the kinetics of the UGC variant of tRNA Val (Figure 2G ). Interestingly, the latter showed a linear increase of k obs as a function of CmoM concentration, indicating that the substrate tRNA was non-optimal ( 45 ) and that a conformational transition of the enzyme-tRNA-AdoMet complex was necessary to adopt an active state. For example, this conformational transition might involve re-arrangement of the anticodon stem-loop of tRNA Val , which differs from that of tRNA Pro and all other CmoM substrates by having a C30–G40 pair instead of a G30–C40 pair ( Supplementary Figure S1 ). The kinetic distinction between the WT tRNA Pro and the active mutant of tRNA Val demonstrates that our single-turnover assays have the ability to discern subtle differences between substrates during methyl transfer.
The saturation kinetics of the WT UGG tRNA Pro showed that the single-turnover parameters for CmoM ( k chem = 0.018 ± 0.003 s −1 ; K d (tRNA) = 5.3 ± 1.9 μM, and k chem / K d = 0.0034 ± 0.0013 s −1 μM −1 , Figure 2H ) are well within the range of other AdoMet-dependent tRNA methyl transferases ( 27 , 29 , 38–40 ). For the UGC variant of tRNA Val , because partial saturation kinetics began to appear at 8 μM of the enzyme, fitting the data to a hyperbolic equation showed k chem ∼ 0.66 s −1 , K d ∼ 26 μM, and k chem / K d ∼ 0.025 s −1 μM −1 (not shown). To independently validate the kinetics, we performed single-turnover assays to measure k chem / K d under conditions where the enzyme was sub- K d such that the k obs was a close representation of the efficiency. Kinetic analysis with the enzyme at 1 μM (∼25-fold below the K d ) showed the k chem / K d of 0.024 ± 0.003 s −1 μM −1 , Figure 2H ), closely similar to the estimated value from the hyperbolic fit. This result reveals an increase of almost 7-fold efficiency for the mutant tRNA Val relative to the WT tRNA Pro (Figure 2H ) and is consistent with the faster kinetics of the mutant tRNA to reach the plateau (Figure 2E ). Thus, once the initial non-optimal enzyme-tRNA-AdoMet conformation was overcome in the mutant tRNA, the structure of the transformed complex was favorable for high efficiency of methyl transfer in a process driven by G35. These data support the hypothesis that the single G35 base is the major determinant of tRNA recognition by CmoM.
To validate the design principle of our genetic approach, which is particularly useful for tRNA species essential for cell survival, we tested the hypothesis that mutations in an essential tRNA would create cellular toxicity. Using UGG tRNA Pro as an example, where the UGG anticodon is a major determinant of charging of tRNA Pro by ProRS ( 46 ), we showed that the creation of the UAG anticodon mutation, which reads Leu codons CU[G/A], reduced specific charging with Pro to 90% and increased mis-charging with Leu to 10% ( Supplementary Figure S6C ). While charging was assayed in vitro with purified enzymes, the mutant UAG tRNA Pro was purified from E. coli cells lacking only cmoM but retaining genes for all other modification enzymes, indicating that the data obtained from the mutant tRNA should be relevant in vivo . Indeed, growth analysis showed that cells expressing the UAG variant of tRNA Pro had reduced viability relative to cells without it, even with expression of the maintenance UGG- proL gene ( Supplementary Figure S6D, E ). The reduced viability is consistent with the notion that the charged UAG tRNA Pro has delivered Pro to Leu codons CU[G/A], resulting in mis-translation during protein synthesis. This toxicity can only be identified in our genetic approach, where a basal level of viability is maintained, but not in an environment, where cell death occurs due to substitution of the natural UGG anticodon in the chromosome.
Dependence on m 1 G37 for terminal methylation by CmoM
UGG tRNA Pro provides an opportunity to explore the inter-dependent relationship of the modifications at positions 34 and 37. In this tRNA, mcmo 5 U is a complex modification conserved at position 34 among γ-proteobacteria ( 5 ), whereas m 1 G37 is a primary methylation (to the N 1 of guanine) conserved across all bacterial species (both Gram-positive and Gram-negative) ( 47 , 48 ). Here we focused on the relationship between these two modifications. The terminal methylation is not essential for growth and its functional role in UGG tRNA Pro has not been characterized, although it has an effect on reading-frame accuracy for another mcmo 5 U34-containing tRNA (encoded by alaT, alaU and alaV , Supplementary Figure S1 ) ( 5 ). In contrast, the m 1 G37 methylation is the major determinant in UGG tRNA Pro for reading-frame accuracy and its elimination substantially increases the frequency of ribosomal shifts in both enzyme- and cell-based assays ( 24 , 32 ). Given that the two types of methylation occur in distinct chemical structure and space with distinct evolutionary distribution, we addressed the question of whether they are independent of each other or whether one is dependent on the other. Answering this question would shed light on the development of a functional UGG tRNA Pro .
We showed that the presence of m 1 G37 affected the terminal methylation level by comparing CmoM activity on UGG tRNA Pro isolated with and without the primary methylation. The tRNA with m 1 G37 was isolated from the E. coli vehicle strain described above, whereas that without m 1 G37 was isolated from a derivative of the vehicle strain that also lacked the trmD gene. Because trmD is essential for growth ( 24 ), a simple knockout cannot be made. In the derivative, the chromosomal trmD was eliminated, but cell viability was maintained by the Ara-controlled expression of the human counterpart trm5 ( 40 ) from the pACYC maintenance plasmid ( Supplementary Figure S7 ) ( 24 ). Cells were grown to saturation in the presence of Ara to maintain the activity of trm5 and 1% of these cells were then transferred to fresh media without Ara and grown for 3 h, followed by another transfer of 20% cells to fresh media and growth for 2 h. This serial dilution turned off trm5 and depleted cellular m 1 G37-tRNA to less than 5% of total tRNA ( 49 ) before isolation of UGG tRNA Pro .
Single-turnover kinetic analysis of CmoM showed a 5-fold reduction in k chem for the tRNA lacking m 1 G37 versus with m 1 G37 (Figure 3A , B ), indicating a 5-fold reduction in the number of enzyme molecules engaged in methyl transfer per unit of time. Thus, the presence of m 1 G37 is important for the action of CmoM. Although the tRNA lacking m 1 G37 had a 6-fold more favorable K d for the enzyme, resulting in an overall k chem / K d similar to that of the control, we note that the driver for the overall level of methylation is determined by k c hem in the context of a cell. This was based on the observation that the CmoM methylation to UGG tRNA Pro progressively increases to the highest level in the stationary phase ( 5 ). The importance of m 1 G37 for the level of terminal methylation at U34 by CmoM is supported by the crystal structure of the ribosomal 30S subunit with the anticodon–stem–loop of tRNA Pro ( 50 ), showing that the anticodon loop would be disordered without m 1 G37 but become organized when the methylation was in place. This ordering and organization of the anticodon loop helped to align the three anticodon bases to properly pair with the cognate codon. The dependence on m 1 G37 for the action of CmoM suggests that the organization of the anticodon by the primary methylation enables the wobble base better recognized for the terminal methylation.
Conversely, we showed that the terminal methylation in mcmo 5 U34 was unaffected by m 1 G37. For broader impact, we explored not only the terminal methylation but also the two precursor states of the wobble modification. Using the trmD-KO strain that we constructed ( 24 ), which provided tRNA lacking m 1 G37 for analysis of TrmD methylation, we isolated UGG tRNA Pro from cells with the wobble base in three different states: the ho 5 U34-state was isolated from the strain with additional cmoB-KO , the cmo 5 U34-state from the strain with additional cmoM-KO , and the mcmo 5 U34-state from the trmD-KO strain. Because the tRNA sequence in each case had no mutation, each was purified by over-expression of the wt gene from the pKK223–3 plasmid in the trmD-KO strain harboring additional gene-knockout of choice. The modification status of each state was previously confirmed by LC-MS/MS in the strain expressing trmD ( Supplementary Figure S6B ). Kinetic analysis of TrmD was performed in a steady-state multi-turnover condition. This was appropriate for TrmD, which unlike CmoM is a non-Rossmann-fold enzyme and is rate-limited at the chemistry of methyl transfer ( 38 ). Because slow chemistry caused slow product release, steady-state assays that monitored product release ( 38 ) would faithfully report the chemistry events of TrmD. For the fully modified mcmo 5 U34-state, the TrmD kinetic parameters k cat (0.29 ± 0.01 s −1 ) and K m (2.7 ± 0.1 μM) (Figure 3C ) are within a 2-fold range of those previously published for the unmodified transcript of the tRNA ( 38 ), indicating that post-transcriptional modifications are not critical for TrmD. Indeed, the k cat , K m and k cat / K m values for all three states of the tRNA were similar with each other (Figure 3C ), supporting the notion that the TrmD activity does not depend on the status of the wobble modification.
Our results establish a hierarchical ranking of the TrmD-catalyzed m 1 G37 over the modification state at the wobble base in UGG tRNA Pro (Figure 3D ). While we only studied the effect of m 1 G37 on the terminal methylation of the wobble base, we suggest that the effect would be maintained throughout all states of the wobble modification, because the primary methylation is required to organize the anticodon structure to permit each modification step. Thus, m 1 G37 is important not only for reading-frame accuracy of the tRNA, but also for initiating a complex modification process to the wobble base to improve the decoding quality of the latter. This new finding raises the possibility that the maturation of a functional UGG tRNA Pro must have acquired m 1 G37 before mcmo 5 U34. Without the former to maintain the reading frame and cell survival ( 24 ), the latter would not form and the quality of tRNA decoding would be poor. Thus, the priority placement of m 1 G37 over mcmo 5 U34 has biological significance in that tRNA must support cell growth before it improves the decoding quality.
The interdependent relationship between tRNA positions 34 and 37 has been probed for two other cases. In one, the formation of C m 32 (2′- O -methylation of C32) and G m 34 (2′- O -methylation of G34) in tRNA Phe is found to drive the conversion of m 1 G37 to yW (wybutosine) ( 51 ). In the second, the synthesis of t 6 A37 (threonylcarbamoyl adenosine) in tRNA Ile is found to precede before aminoacylation by IleRS and possibly lysidinylation of C34 by TilS ( 52 ). The hierarchical ranking of the first case, placing positions 32 and 34 over 37, is opposite from our finding, whereas that of the second, placing position 37 over 34, is similar. To determine if there are rules governing the hierarchy, further studies are needed. For example, four other tRNA families with cmo 5 U34 or mcmo 5 U34 contain a different modification at position 37 ( Supplementary Figure S1 ). These are m 6 A37 ( N 6 -methyl-A37) for UAC tRNA Val ( 53 ), ms 2 i 6 A37 (2-methylthio- N 6 -isopentenyl-A37) for UGA tRNA Ser ( 54 , 55 ), ct 6 A37 (cyclic N 6 -threonylcarbamoyl-A37) for UGU tRNA Thr ( 56 , 57 ), and m 1 G37 for UAG tRNA Leu ( 20 ). Little is known about the hierarchical relationship between the two positions in the development of each tRNA. Based on our work here, we suggest that each tRNA has a priority placement of modifications between positions 34 and 37 to ensure the functional state of the tRNA in the context of a cell.
Supplementary Material
Click here for additional data file.
ACKNOWLEDGEMENTS
We thank Dr. Steven Almo for the E. coli cmoB-KO strain.
SUPPLEMENTARY DATA
Supplementary data are available at NAR online.
FUNDING
NIH [GM1081601 and GM114343 to Y.M.H.]; JSPS postdoctoral fellowship (to I.M.); Defense Threat Reduction Agency HDTRA [1-15-1-0033 to P.L.]. Funding for open access charge: NIH [GM081601 to Y.M.H., GM114343 to Y.M.H.].
Conflict of interest statement . None declared.
REFERENCES
1. Suzuki T. Grosjean H 2005 ; 12 : Springer-Verlag Berlin and Heidelberg, GmbH & Co 23 – 69 .
2. Machnicka M.A. , Milanowska K. , Osman Oglou O. , Purta E. , Kurkowska M. , Olchowik A. , Januszewski W. , Kalinowski S. , Dunin-Horkawicz S. , Rother K.M. MODOMICS: a database of RNA modification pathways–2013 update . Nucleic Acids Res. 2013 ; 41 : D262 – D267 . 23118484 10.1093/nar/gks1007 PMC3531130
3. Murao K. , Saneyoshi M. , Harada F. , Nishimura S. Uridin-5-oxy acetic acid: a new minor constituent from E. coli valine transfer RNA I . Biochem. Biophys. Res. Commun. 1970 ; 38 : 657 – 662 . 4910247 10.1016/0006-291x(70)90631-5
4. Sorensen M.A. , Elf J. , Bouakaz E. , Tenson T. , Sanyal S. , Bjork G.R. , Ehrenberg M. Over expression of a tRNA Leu isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading . J Mol Biol . 2005 ; 354 : 16 – 24 . 16236318 10.1016/j.jmb.2005.08.076
5. Sakai Y. , Miyauchi K. , Kimura S. , Suzuki T. Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons . Nucleic Acids Res. 2016 ; 44 : 509 – 523 . 26681692 10.1093/nar/gkv1470 PMC4737166
6. Osawa S. , Jukes T.H. , Watanabe K. , Muto A. Recent evidence for evolution of the genetic code . Microbiol. Rev. 1992 ; 56 : 229 – 264 . 1579111 10.1128/mr.56.1.229-264.1992 PMC372862
7. Grosjean H. , de Crecy-Lagard V. , Marck C. Deciphering synonymous codons in the three domains of life: co-evolution with specific tRNA modification enzymes . FEBS Lett. 2010 ; 584 : 252 – 264 . 19931533 10.1016/j.febslet.2009.11.052
8. Lagerkvist U. Unconventional methods in codon reading . Bioessays . 1986 ; 4 : 223 – 226 . 3790123 10.1002/bies.950040509
9. Rogalski M. , Karcher D. , Bock R. Superwobbling facilitates translation with reduced tRNA sets . Nat. Struct. Mol. Biol. 2008 ; 15 : 192 – 198 . 18193063 10.1038/nsmb.1370
10. Claesson C. , Lustig F. , Boren T. , Simonsson C. , Barciszewska M. , Lagerkvist U. Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop . J. Mol. Biol. 1995 ; 247 : 191 – 196 . 7707368 10.1006/jmbi.1994.0132
11. Takai K. , Takaku H. , Yokoyama S. Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis . Nucleic Acids Res. 1996 ; 24 : 2894 – 2899 . 8760870 10.1093/nar/24.15.2894 PMC146040
12. Phelps S.S. , Malkiewicz A. , Agris P.F. , Joseph S. Modified nucleotides in tRNA Lys and tRNA Val are important for translocation . J. Mol. Biol. 2004 ; 338 : 439 – 444 . 15081802 10.1016/j.jmb.2004.02.070
13. Samuelsson T. , Elias P. , Lustig F. , Axberg T. , Folsch G. , Akesson B. , Lagerkvist U. Aberrations of the classic codon reading scheme during protein synthesis in vitro . J. Biol. Chem. 1980 ; 255 : 4583 – 4588 . 6989813
14. Weixlbaumer A. , Murphy F.V.t. , Dziergowska A. , Malkiewicz A. , Vendeix F.A. , Agris P.F. , Ramakrishnan V. Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines . Nat. Struct. Mol. Biol. 2007 ; 14 : 498 – 502 . 17496902 10.1038/nsmb1242 PMC2816034
15. Nasvall S.J. , Chen P. , Bjork G.R. The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo 5 UGG) promotes reading of all four proline codons in vivo . RNA . 2004 ; 10 : 1662 – 1673 . 15383682 10.1261/rna.7106404 PMC1370651
16. Yokoyama S. , Watanabe T. , Murao K. , Ishikura H. , Yamaizumi Z. , Nishimura S. , Miyazawa T. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon . Proc. Natl. Acad. Sci. U.S.A. 1985 ; 82 : 4905 – 4909 . 3860833 10.1073/pnas.82.15.4905 PMC390466
17. Kim J. , Xiao H. , Bonanno J.B. , Kalyanaraman C. , Brown S. , Tang X. , Al-Obaidi N.F. , Patskovsky Y. , Babbitt P.C. , Jacobson M.P. Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function . Nature . 2013 ; 498 : 123 – 126 . 23676670 10.1038/nature12180 PMC3895326
18. Bjork G.R. A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli . J. Mol. Biol. 1980 ; 140 : 391 – 410 . 6160251 10.1016/0022-2836(80)90391-5
19. Hagervall T.G. , Jonsson Y.H. , Edmonds C.G. , McCloskey J.A. , Bjork G.R. Chorismic acid, a key metabolite in modification of tRNA . J. Bacteriol. 1990 ; 172 : 252 – 259 . 2104604 10.1128/jb.172.1.252-259.1990 PMC208425
20. Qian Q. , Bjork G.R. Structural requirements for the formation of 1-methylguanosine in vivo in tRNA(Pro)GGG of Salmonella typhimurium . J. Mol. Biol. 1997 ; 266 : 283 – 296 . 9047363 10.1006/jmbi.1996.0789
21. Hou Y.M. , Schimmel P. A simple structural feature is a major determinant of the identity of a transfer RNA . Nature . 1988 ; 333 : 140 – 145 . 3285220 10.1038/333140a0
22. Hou Y.M. , Schimmel P. Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution . Biochemistry . 1989 ; 28 : 6800 – 6804 . 2684266 10.1021/bi00443a003
23. Hou Y.M. , Motegi H. , Lipman R.S. , Hamann C.S. , Shiba K. Conservation of a tRNA core for aminoacylation . Nucleic Acids Res. 1999 ; 27 : 4743 – 4750 . 10572174 10.1093/nar/27.24.4743 PMC148774
24. Gamper H.B. , Masuda I. , Frenkel-Morgenstern M. , Hou Y.M. Maintenance of protein synthesis reading frame by EF-P and m 1 G37-tRNA . Nat. Commun. 2015 ; 6 : 7226 . 26009254 10.1038/ncomms8226 PMC4445466
25. Cherepanov P.P. , Wackernagel W. Gene disruption in Escherichia coli : Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant . Gene . 1995 ; 158 : 9 – 14 . 7789817 10.1016/0378-1119(95)00193-a
26. Datsenko K.A. , Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products . Proc. Natl. Acad. Sci. U.S.A. 2000 ; 97 : 6640 – 6645 . 10829079 10.1073/pnas.120163297 PMC18686
27. Christian T. , Sakaguchi R. , Perlinska A.P. , Lahoud G. , Ito T. , Taylor E.A. , Yokoyama S. , Sulkowska J.I. , Hou Y.M. Methyl transfer by substrate signaling from a knotted protein fold . Nat. Struct. Mol. Biol. 2016 ; 23 : 941 – 948 . 27571175 10.1038/nsmb.3282 PMC5429141
28. Christian T. , Evilia C. , Williams S. , Hou Y.M. Distinct origins of tRNA(m 1 G37) methyltransferase . J. Mol. Biol. 2004 ; 339 : 707 – 719 . 15165845 10.1016/j.jmb.2004.04.025
29. Christian T. , Hou Y.M. Distinct determinants of tRNA recognition by the TrmD and Trm5 methyl transferases . J. Mol. Biol. 2007 ; 373 : 623 – 632 . 17868690 10.1016/j.jmb.2007.08.010 PMC2064070
30. Zhang C.M. , Perona J.J. , Ryu K. , Francklyn C. , Hou Y.M. Distinct kinetic mechanisms of the two classes of aminoacyl-tRNA synthetases . J. Mol. Biol. 2006 ; 361 : 300 – 311 . 16843487 10.1016/j.jmb.2006.06.015
31. Liu C. , Gamper H. , Liu H. , Cooperman B.S. , Hou Y.M. Potential for interdependent development of tRNA determinants for aminoacylation and ribosome decoding . Nat. Commun. 2011 ; 2 : 329 . 21629262 10.1038/ncomms1331 PMC3799875
32. Gamper H.B. , Masuda I. , Frenkel-Morgenstern M. , Hou Y.M. The UGG isoacceptor of tRNA Pro is naturally prone to frameshifts . Int. J. Mol. Sci. 2015 ; 16 : 14866 – 14883 . 26140378 10.3390/ijms160714866 PMC4519876
33. Lahoud G. , Goto-Ito S. , Yoshida K. , Ito T. , Yokoyama S. , Hou Y.M. Differentiating analogous tRNA methyltransferases by fragments of the methyl donor . RNA . 2011 ; 17 : 1236 – 1246 . 21602303 10.1261/rna.2706011 PMC3138561
34. Sakaguchi R. , Giessing A. , Dai Q. , Lahoud G. , Liutkeviciute Z. , Klimasauskas S. , Piccirilli J. , Kirpekar F. , Hou Y.M. Recognition of guanosine by dissimilar tRNA methyltransferases . RNA . 2012 ; 18 : 1687 – 1701 . 22847817 10.1261/rna.032029.111 PMC3425783
35. Sakaguchi R. , Lahoud G. , Christian T. , Gamper H. , Hou Y.M. A divalent metal ion-dependent N 1 -methyl transfer to G37-tRNA . Chem. Biol. 2014 ; 21 : 1351 – 1360 . 25219964 10.1016/j.chembiol.2014.07.023 PMC4224600
36. Naganuma M. , Sekine S. , Chong Y.E. , Guo M. , Yang X.L. , Gamper H. , Hou Y.M. , Schimmel P. , Yokoyama S. The selective tRNA aminoacylation mechanism based on a single G*U pair . Nature . 2014 ; 510 : 507 – 511 . 24919148 10.1038/nature13440 PMC4323281
37. Hamann C.S. , Hou Y.M. An RNA structural determinant for tRNA recognition . Biochemistry . 1997 ; 36 : 7967 – 7972 . 9201943 10.1021/bi970517w
38. Christian T. , Lahoud G. , Liu C. , Hou Y.M. Control of catalytic cycle by a pair of analogous tRNA modification enzymes . J. Mol. Biol. 2010 ; 400 : 204 – 217 . 20452364 10.1016/j.jmb.2010.05.003 PMC2892103
39. Christian T. , Lahoud G. , Liu C. , Hoffmann K. , Perona J.J. , Hou Y.M. Mechanism of N-methylation by the tRNA m 1 G37 methyltransferase Trm5 . RNA . 2010 ; 16 : 2484 – 2492 . 20980671 10.1261/rna.2376210 PMC2995409
40. Christian T. , Gamper H. , Hou Y.M. Conservation of structure and mechanism by Trm5 enzymes . RNA . 2013 ; 19 : 1192 – 1199 . 23887145 10.1261/rna.039503.113 PMC3753926
41. Schubert H.L. , Blumenthal R.M. , Cheng X. Many paths to methyltransfer: a chronicle of convergence . Trends Biochem. Sci. 2003 ; 28 : 329 – 335 . 12826405 10.1016/S0968-0004(03)00090-2 PMC2758044
42. Martin J.L. , McMillan F.M. SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold . Curr. Opin. Struct. Biol. 2002 ; 12 : 783 – 793 . 12504684 10.1016/s0959-440x(02)00391-3
43. Liu C. , Gamper H. , Shtivelband S. , Hauenstein S. , Perona J.J. , Hou Y.M. Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase . J. Mol. Biol. 2007 ; 367 : 1063 – 1078 . 17303165 10.1016/j.jmb.2007.01.050 PMC2041962
44. Christian T. , Evilia C. , Hou Y.M. Catalysis by the second class of tRNA(m 1 G37) methyl transferase requires a conserved proline . Biochemistry . 2006 ; 45 : 7463 – 7473 . 16768442 10.1021/bi0602314 PMC2517134
45. Dupasquier M. , Kim S. , Halkidis K. , Gamper H. , Hou Y.M. tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control . J. Mol. Biol. 2008 ; 379 : 579 – 588 . 18466919 10.1016/j.jmb.2008.04.005 PMC2430420
46. Cusack S. , Yaremchuk A. , Krikliviy I. , Tukalo M. tRNA Pro anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase . Structure . 1998 ; 6 : 101 – 108 . 9493271 10.1016/s0969-2126(98)00011-2
47. Bjork G.R. , Jacobsson K. , Nilsson K. , Johansson M.J. , Bystrom A.S. , Persson O.P. A primordial tRNA modification required for the evolution of life? . EMBO J. 2001 ; 20 : 231 – 239 . 11226173 10.1093/emboj/20.1.231 PMC140193
48. Bystrom A.S. , Bjork G.R. The structural gene ( trmD ) for the tRNA(m 1 G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-12 . Mol. Gen. Genet. 1982 ; 188 : 447 – 454 . 6298574 10.1007/BF00330047
49. Masuda I. , Sakaguchi R. , Liu C. , Gamper H. , Hou Y.M. The temperature sensitivity of a mutation in the essential tRNA modification enzyme tRNA methyltransferase D (TrmD) . J. Biol. Chem. 2013 ; 288 : 28987 – 28996 . 23986443 10.1074/jbc.M113.485797 PMC3789996
50. Maehigashi T. , Dunkle J.A. , Miles S.J. , Dunham C.M. Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops . Proc. Natl. Acad. Sci. U.S.A. 2014 ; 111 : 12740 – 12745 . 25128388 10.1073/pnas.1409436111 PMC4156745
51. Guy M.P. , Podyma B.M. , Preston M.A. , Shaheen H.H. , Krivos K.L. , Limbach P.A. , Hopper A.K. , Phizicky E.M. Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNA Phe anticodon loop . RNA . 2012 ; 18 : 1921 – 1933 . 22912484 10.1261/rna.035287.112 PMC3446714
52. Thiaville P.C. , El Yacoubi B. , Kohrer C. , Thiaville J.J. , Deutsch C. , Iwata-Reuyl D. , Bacusmo J.M. , Armengaud J. , Bessho Y. , Wetzel C. Essentiality of threonylcarbamoyladenosine (t 6 A), a universal tRNA modification, in bacteria . Mol. Microbiol. 2015 ; 98 : 1199 – 1221 . 26337258 10.1111/mmi.13209 PMC4963248
53. Golovina A.Y. , Sergiev P.V. , Golovin A.V. , Serebryakova M.V. , Demina I. , Govorun V.M. , Dontsova O.A. The yfiC gene of E. coli encodes an adenine-N 6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo 5 UAC) . RNA . 2009 ; 15 : 1134 – 1141 . 19383770 10.1261/rna.1494409 PMC2685529
54. Schweizer U. , Bohleber S. , Fradejas-Villar N. The modified base isopentenyladenosine and its derivatives in tRNA . RNA Biol. 2017 ; 14 : 1 – 12 . 28277934 10.1080/15476286.2017.1294309 PMC5699536
55. Motorin Y. , Bec G. , Tewari R. , Grosjean H. Transfer RNA recognition by the Escherichia coli delta2-isopentenyl-pyrophosphate:tRNA delta2-isopentenyl transferase: dependence on the anticodon arm structure . RNA . 1997 ; 3 : 721 – 733 . 9214656 PMC1369520
56. Miyauchi K. , Kimura S. , Suzuki T. A cyclic form of N 6 -threonylcarbamoyladenosine as a widely distributed tRNA hypermodification . Nat. Chem. Biol. 2013 ; 9 : 105 – 111 . 23242255 10.1038/nchembio.1137
57. Kimura S. , Miyauchi K. , Ikeuchi Y. , Thiaville P.C. , Crecy-Lagard V. , Suzuki T. Discovery of the beta-barrel-type RNA methyltransferase responsible for N 6 -methylation of N 6 -threonylcarbamoyladenosine in tRNAs . Nucleic Acids Res. 2014 ; 42 : 9350 – 9365 . 25063302 10.1093/nar/gku618 PMC4132733