Anticodon Loop Modification Research Articles

Posttranscriptional modification to the core of tRNAs modulates translational misreading errors.

Protein synthesis on the ribosome involves successive rapid recruitment of cognate aminoacyl-tRNAs and rejection of the much more numerous incorrect near- or non-cognates. The principal feature of translation elongation is that at every step, many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translational accuracy require that cognate tRNAs have relatively similar acceptance rates by the ribosome. To achieve that, tRNAs evolved to compensate for differences in amino acid properties and codon-anticodon strength that affect acceptance. Part of that response involved tRNA posttranscriptional modifications, which can affect tRNA decoding efficiency, accuracy, and structural stability. The most intensively modified regions of the tRNA are the anticodon loop and structural core of the tRNA. Anticodon loop modifications directly affect codon-anticodon pairing and therefore modulate accuracy. Core modifications have been thought to ensure consistent decoding rates principally by stabilizing tRNA structure to avoid degradation; however, degradation due to instability appears to only be a significant issue above normal growth temperatures. We suspected that the greater role of modification at normal temperatures might be to tune tRNAs to maintain consistent intrinsic rates of acceptance and peptide transfer and that hypomodification by altering these rates might degrade the process of discrimination, leading to increased translational errors. Here, we present evidence that most tRNA core modifications do modulate the frequency of misreading errors, suggesting that the need to maintain accuracy explains their deep evolutionary conservation.

Abstract PR001: Codon-bias and tRNA modifications as drivers of melanoma metastasis

Abstract Only tumor cells that are able to enhance their stress response pathways, are able to successfully metastasize and colonize distant organs. In yeast, reprogramming of translation through modification of the tRNA anticodon loop in response to stress has been shown to be a central adaptive mechanism of survival. Distinct tRNA modifications increase translational efficiency of certain codons. These modifications are upregulated and are essential for survival under a range of stressors, including oxidative stress. The adaptive proteome is therefore encoded by specific transcripts with a distinct codon bias and regulated by these tRNA modifications. We have identified several stress-induced tRNA anticodon loop modifications in metastasizing melanoma cells that drive codon-biased translation of the adaptive pro-metastatic proteome. One of these modifications specifically regulates stress-induced translation of selenocysteine-containing proteins. Selenoproteins are central to stress resistance including oxidative and proteotoxic stress. We have identified and characterized the methyltransferase that regulates this modification and enables translation of the metastatic selenoproteome. More broadly, by combining biochemical approaches with in vivo models of melanoma metastasis, we have now identified several other enzymes responsible for multiple tRNA anticodon modifications as novel and targetable drivers of metastatic disease. Our work aims to establish how tRNA anticodon modifications and a distinct codon bias reprogram the metastatic proteome as a therapeutically targetable stress response mechanism. Citation Format: Elena Piskounova. Codon-bias and tRNA modifications as drivers of melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR001.

"Superwobbling" and tRNA-34 Wobble and tRNA-37 Anticodon Loop Modifications in Evolution and Devolution of the Genetic Code.

The genetic code evolved around the reading of the tRNA anticodon on the primitive ribosome, and tRNA-34 wobble and tRNA-37 modifications coevolved with the code. We posit that EF-Tu, the closing mechanism of the 30S ribosomal subunit, methylation of wobble U34 at the 5-carbon and suppression of wobbling at the tRNA-36 position were partly redundant and overlapping functions that coevolved to establish the code. The genetic code devolved in evolution of mitochondria to reduce the size of the tRNAome (all of the tRNAs of an organism or organelle). “Superwobbling” or four-way wobbling describes a major mechanism for shrinking the mitochondrial tRNAome. In superwobbling, unmodified wobble tRNA-U34 can recognize all four codon wobble bases (A, G, C and U), allowing a single unmodified tRNA-U34 to read a 4-codon box. During code evolution, to suppress superwobbling in 2-codon sectors, U34 modification by methylation at the 5-carbon position appears essential. As expected, at the base of code evolution, tRNA-37 modifications mostly related to the identity of the adjacent tRNA-36 base. TRNA-37 modifications help maintain the translation frame during elongation.

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications.

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.

Unfolded Protein Response Suppression in Yeast by Loss of tRNA Modifications.

Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyze whether this includes the activation of the unfolded protein response (UPR), which is commonly triggered by protein aggregation within the endoplasmic reticulum (ER). We demonstrate that two different aggregation prone tRNA modification mutants (elp6 ncs2; elp3 deg1) lacking combinations of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U: elp3; elp6; ncs2) and pseudouridine (Ψ: deg1) reduce, rather than increase, splicing of HAC1 mRNA, an event normally occurring as a precondition of UPR induction. In addition, tunicamycin (TM) induced HAC1 splicing is strongly impaired in the elp3 deg1 mutant. Strikingly, this mutant displays UPR independent resistance against TM, a phenotype we found to be rescued by overexpression of tRNAGln(UUG), the tRNA species usually carrying the mcm5s2U34 and Ψ38 modifications. Our data indicate that proper tRNA anticodon loop modifications promote rather than impair UPR activation and reveal that protein synthesis and homeostasis defects in their absence do not routinely result in UPR induction but may relieve endogenous ER stress.

Lack of 2'-O-methylation in the tRNA anticodon loop of two phylogenetically distant yeast species activates the general amino acid control pathway.

Modification defects in the tRNA anticodon loop often impair yeast growth and cause human disease. In the budding yeast Saccharomyces cerevisiae and the phylogenetically distant fission yeast Schizosaccharomyces pombe, trm7Δ mutants grow poorly due to lack of 2'-O-methylation of C32 and G34 in the tRNAPhe anticodon loop, and lesions in the human TRM7 homolog FTSJ1 cause non-syndromic X-linked intellectual disability (NSXLID). However, it is unclear why trm7Δ mutants grow poorly. We show here that despite the fact that S. cerevisiae trm7Δ mutants had no detectable tRNAPhe charging defect in rich media, the cells constitutively activated a robust general amino acid control (GAAC) response, acting through Gcn2, which senses uncharged tRNA. Consistent with reduced available charged tRNAPhe, the trm7Δ growth defect was suppressed by spontaneous mutations in phenylalanyl-tRNA synthetase (PheRS) or in the pol III negative regulator MAF1, and by overexpression of tRNAPhe, PheRS, or EF-1A; all of these also reduced GAAC activation. Genetic analysis also demonstrated that the trm7Δ growth defect was due to the constitutive robust GAAC activation as well as to the reduced available charged tRNAPhe. Robust GAAC activation was not observed with several other anticodon loop modification mutants. Analysis of S. pombe trm7 mutants led to similar observations. S. pombe Trm7 depletion also resulted in no observable tRNAPhe charging defect and a robust GAAC response, and suppressors mapped to PheRS and reduced GAAC activation. We speculate that GAAC activation is widely conserved in trm7 mutants in eukaryotes, including metazoans, and might play a role in FTSJ1-mediated NSXLID.

FactorsThatShapeEukaryotictRNAomes: Processing,ModificationandAnticodon-CodonUse.

Transfer RNAs (tRNAs) contain sequence diversity beyond their anticodons and the large variety of nucleotide modifications found in all kingdoms of life. Some modifications stabilize structure and fit in the ribosome whereas those to the anticodon loop modulate messenger RNA (mRNA) decoding activity more directly. The identities of tRNAs with some universal anticodon loop modifications vary among distant and parallel species, likely to accommodate fine tuning for their translation systems. This plasticity in positions 34 (wobble) and 37 is reflected in codon use bias. Here, we review convergent evidence that suggest that expansion of the eukaryotic tRNAome was supported by its dedicated RNA polymerase III transcription system and coupling to the precursor-tRNA chaperone, La protein. We also review aspects of eukaryotic tRNAome evolution involving G34/A34 anticodon-sparing, relation to A34 modification to inosine, biased codon use and regulatory information in the redundancy (synonymous) component of the genetic code. We then review interdependent anticodon loop modifications involving position 37 in eukaryotes. This includes the eukaryote-specific tRNA modification, 3-methylcytidine-32 (m3C32) and the responsible gene, TRM140 and homologs which were duplicated and subspecialized for isoacceptor-specific substrates and dependence on i6A37 or t6A37. The genetics of tRNA function is relevant to health directly and as disease modifiers.

Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications

ABSTRACTRecently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm5U or s2U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNALysUUU dependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm5U and s2U leads to increased ribosome pausing at tRNALysUUU dependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm5U and s2U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNALysUUU. Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t6A) to its cyclic derivative (ct6A) at the anticodon adjacent position 37. Since loss of ct6A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t6A to ct6A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.

TRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Defects in tRNA Anticodon Loop 2′-O-Methylation Are Implicated in Nonsyndromic X-Linked Intellectual Disability due to Mutations inFTSJ1

tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734, respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause nonsyndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here, we report that tRNA(Phe) from two genetically independent cell lines of NSXLID patients with loss-of-function FTSJ1 mutations nearly completely lacks Cm32 and Gm34 , and has reduced peroxywybutosine (o2yW37 ). Additionally, tRNA(Phe) from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34 , but has normal levels of Cm32 and o2yW37 . tRNA(Phe) from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34 , and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNA(Phe) as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNA(Phe) .

Conservation of an intricate circuit for crucial modifications of the tRNAPhe anticodon loop in eukaryotes.

Post-transcriptional tRNA modifications are critical for efficient and accurate translation, and have multiple different roles. Lack of modifications often leads to different biological consequences in different organisms, and in humans is frequently associated with neurological disorders. We investigate here the conservation of a unique circuitry for anticodon loop modification required for healthy growth in the yeast Saccharomyces cerevisiae. S. cerevisiae Trm7 interacts separately with Trm732 and Trm734 to 2′-O-methylate three substrate tRNAs at anticodon loop residues C32 and N34, and these modifications are required for efficient wybutosine formation at m1G37 of tRNAPhe. Moreover, trm7Δ and trm732Δ trm734Δ mutants grow poorly due to lack of functional tRNAPhe. It is unknown if this circuitry is conserved and important for tRNAPhe modification in other eukaryotes, but a likely human TRM7 ortholog is implicated in nonsyndromic X-linked intellectual disability. We find that the distantly related yeast Schizosaccharomyces pombe has retained this circuitry for anticodon loop modification, that S. pombe trm7Δ and trm734Δ mutants have more severe phenotypes than the S. cerevisiae mutants, and that tRNAPhe is the major biological target. Furthermore, we provide evidence that Trm7 and Trm732 function is widely conserved throughout eukaryotes, since human FTSJ1 and THADA, respectively, complement growth defects of S. cerevisiae trm7Δ and trm732Δ trm734Δ mutants by modifying C32 of tRNAPhe, each working with the corresponding S. cerevisiae partner protein. These results suggest widespread importance of 2′-O-methylation of the tRNA anticodon loop, implicate tRNAPhe as the crucial substrate, and suggest that this modification circuitry is important for human neuronal development.

Different types of secondary information in the genetic code.

Whole-genome and functional analyses suggest a wealth of secondary or auxiliary genetic information (AGI) within the redundancy component of the genetic code. Although there are multiple aspects of biased codon use, we focus on two types of auxiliary information: codon-specific translational pauses that can be used by particular proteins toward their unique folding and biased codon patterns shared by groups of functionally related mRNAs with coordinate regulation. AGI is important to genetics in general and to human disease; here, we consider influences of its three major components, biased codon use itself, variations in the tRNAome, and anticodon modifications that distinguish synonymous decoding. AGI is plastic and can be used by different species to different extents, with tissue-specificity and in stress responses. Because AGI is species-specific, it is important to consider codon-sensitive experiments when using heterologous systems; for this we focus on the tRNA anticodon loop modification enzyme, CDKAL1, and its link to type 2 diabetes. Newly uncovered tRNAome variability among humans suggests roles in penetrance and as a genetic modifier and disease modifier. Development of experimental and bioinformatics methods are needed to uncover additional means of auxiliary genetic information.

Human tRNASecassociates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers

We have shown previously that simple RNA structures bind pure phospholipid liposomes. However, binding of bona fide cellular RNAs under physiological ionic conditions is shown here for the first time. Human tRNA(Sec) contains a hydrophobic anticodon-loop modification: N⁶-isopentenyladenosine (i⁶A) adjacent to its anticodon. Using a highly specific double-probe hybridization assay, we show mature human tRNA(Sec) specifically retained in HeLa intermediate-density membranes. Further, isolated human tRNA(Sec) rebinds to liposomes from isolated HeLa membrane lipids, to a much greater extent than an unmodified tRNA(Sec) transcript. To better define this affinity, experiments with pure lipids show that liposomes forming rafts or including positively charged sphingosine, or particularly both together, exhibit increased tRNA(Sec) binding. Thus tRNA(Sec) residence on membranes is determined by several factors, such as hydrophobic modification (likely isopentenylation of tRNA(Sec)), lipid structure (particularly lipid rafts), or sphingosine at a physiological concentration in rafted membranes. From prior work, RNA structure and ionic conditions also appear important. tRNA(Sec) dissociation from HeLa liposomes implies a mean membrane residence of 7.6 min at 24°C (t(1/2) = 5.3 min). Clearly RNA with a 5-carbon hydrophobic modification binds HeLa membranes, probably favoring raft domains containing specific lipids, for times sufficient to alter biological fates.

Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2'-O-methylation of C(32) and N(34) of tRNA(Phe), tRNA(Trp), and tRNA(Leu(UAA)) to form Cm(32) and Nm(34), and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA(Phe) suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA(Phe). We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2'-O-methylation of C(32), and with Rtt10 (named Trm734) for 2'-O-methylation of N(34) of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA(Phe), in which formation of Cm(32) and Gm(34) drives modification of m(1)G(37) (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm(32) and Nm(34), and the accompanying loss of yW, because trm732-Δ trm734-Δ mutants phenocopy trm7-Δ mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-Δ mutants, which lack m(2,2)G(26) in their tRNAs. We speculate that 2'-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

Anticodon loop modifications of E. coli tRNAArgICG modulate structural flexibility and restrict decoding of the rare CGA codon

Six codons are decoded by tRNAArg in E. coli, and three (CGU, CGC and CGA) are read by tRNAArg1,2isoacceptors. The anticodon stem and loop (ASL) domains of these isoacceptors differ only in the identity of the residue at position 32 as either a 2-thiocytosine (s2C32) or unmodified cytosine for tRNAArg1 and tRNAArg2 respectively. These isoacceptors also contain other naturally-occurring modifications at positions 34 (inosine, I) and 37 (2-methyladenosine, m2A37). To investigate the roles of these modifications in proper folding of the ASL, six ASL constructs, differing in their array of modifications, were analyzed by biophysical spectroscopic methods as well as functional binding assays. Although thermal denaturation and circular dichroism spectroscopy showed that the modifications contribute competing thermodynamic and base stacking properties indicating significant differences in structural dynamics, native gel electrophoresis and NMR spectroscopy clearly show that the equilibrium solution conformations of the ASLs are nearly identical, but do not possess the canonical U-turn structure needed for codon binding. Ribosome filter binding assays show that both s2C32 and m2A37 restrict binding to the rare CGA codon. Inosine seems to be the only modification required for cognate codon binding in the ribosomal A-site; therefore, ribosome binding experiments are being performed with the near cognate codons UGU, UGC and UGA to determine if the modifications inhibit misreading. Taken together, the results clearly indicate that chemical modifications modulate the flexibility of the loop; however, it remains unclear how they function in codon recognition. This research is supported by the National Science Foundation.

Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio-N6-threonylcarbamoyladenosine in tRNA

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival

tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm(5)U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm(5)U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm(5)U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.

Tertiary structure checkpoint at anticodon loop modification in tRNA functional maturation

tRNA precursors undergo a maturation process, involving nucleotide modifications and folding into the L-shaped tertiary structure. The N1-methylguanosine at position 37 (m1G37), 3' adjacent to the anticodon, is essential for translational fidelity and efficiency. In archaea and eukaryotes, Trm5 introduces the m1G37 modification into all tRNAs bearing G37. Here we report the crystal structures of archaeal Trm5 (aTrm5) in complex with tRNA(Leu) or tRNA(Cys). The D2-D3 domains of aTrm5 discover and modify G37, independently of the tRNA sequences. D1 is connected to D2-D3 through a flexible linker and is designed to recognize the shape of the tRNA outer corner, as a hallmark of the completed L shape formation. This interaction by D1 lowers the K(m) value for tRNA, enabling the D2-D3 catalysis. Thus, we propose that aTrm5 provides the tertiary structure checkpoint in tRNA maturation.