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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: IUBMB Life. 2019 Jul 8;71(8):1158–1166. doi: 10.1002/iub.2120

Bacterial Wobble Modifications of NNA-Decoding tRNAs

Emil M Nilsson 1, Rebecca W Alexander 1,*
PMCID: PMC6893868  NIHMSID: NIHMS1059951  PMID: 31283100

Abstract

Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A-ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A-ending codons are not read by U34-containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A-ending codons.

tRNA MODIFICATIONS

Base pairing rules established in the context of the DNA double helix generally hold true in RNA, although the greater structural and functional diversity of RNA is facilitated by a wide variety of chemical modifications. Transfer RNA (tRNA) serves a key function in genetic information transfer, accurately deciphering each trinucleotide codon of messenger RNA (mRNA) to produce the encoded polypeptide product. tRNA is the most chemically modified RNA species in any organism, with nucleotide modifications impacting tRNA structure, dynamics, and function. To date over 100 unique RNA modifications have been identified, more than 90 of which are found in tRNA (1–3). On average 17% of tRNA nucleotides are post-transcriptionally modified, more than 10 times the frequency observed in larger RNAs such as ribosomal RNA (rRNA), which is modified at 1–2% (1, 4). Post-transcriptional modifications have been shown to influence translational accuracy by directly regulating base pairing on the ribosome (3, 5), dictating aminoacyl-tRNA synthetase activity (6–9), facilitating peptidyl-tRNA translocation (10), and fine-tuning ribosomal kinetics (11, 12). Post-transcriptional modifications occur throughout tRNA; however, nucleotides in the anticodon loop are particularly highly decorated. Nucleotides N34, the wobble position, and N37, just outside the anticodon, display a larger diversity of modification chemistry than any other tRNA position (1, 4, 13). This review will consider modifications at the N34 wobble position in bacterial tRNAs decoding NNA codons.

NNA DECODING

tRNAs translating NNA codons are typically encoded with a uridine at position 34 (U34), as expected according to Watson-Crick rules, and have the intrinsic potential to decode both NNA and NNG codons by classical wobble pairing (14). Such expanded decoding can be advantageous for an organism, as a single tRNA can read both codons. In some cases, however, codon–anticodon wobble pairing could be detrimental to genetic code fidelity. In either situation organisms have evolved post-transcriptional modification schemes to enforce the correct decoding function of each tRNA (15, 16). The nature of these modifications varies among tRNA isoacceptors and across the domains of life, but the high frequency of U34 modification is conserved.

ESCHERICHIA COLI U34 MODIFICATIONS

There are 16 A-ending trinucleotides in the Universal Genetic Code, of which two are the ochre (UAA) and opal (UGA) stop codons. Escherichia coli encodes 29 U34-containing tRNAs to translate 12 sense codons (highlighted in Fig. 1) (17), leaving two A-ending sense codons to be decoded through adaptation of other tRNAs. All U34-containing E. coli tRNAs are post-transcriptionally modified at the wobble position with a total of five unique species (15). The most frequent modification is uridine 5-oxyacetic acid (cmo5U), which is observed in tRNALeuUAG, tRNAValUAC, tRNASerUGA, tRNAThrUGU, tRNAAlaUGC, and tRNAProUGG (16, 18). Codons read by cmo5U-containing tRNAs are highlighted in orange on the Universal Genetic Code table (Fig. 1). Next, tRNAArgUCG and tRNAGlyUCC both contain a 5-methylaminomethyluridine (mnm5U) to translate codons CGA and GGA (highlighted in green in Fig. 1) (19, 20). tRNAGluUUC and tRNALysUUU also have mnm5U34 but are further differentiated through the addition of a 2-thio group, forming 5-methylaminomethyl-2-thiouridine (mnm5s2U) (reading codons highlighted in blue, Fig. 1) (21). tRNAGln is found with the 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) at position 34 while tRNALeuUAA presents a 5-carboxymethylaminomethyl-2´-O-methyluridine (cmnm5Um) (reading codons highlighted in yellow, Fig. 1) (22, 23). Each category of modification will be addressed below in the context of bacterial translation, with respect to structure of the chemical moiety, the enzymatic pathway, how decoding is facilitated, and evolutionary conservation, if known.

FIG 1.

FIG 1

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The universal genetic code. Codons highlighted in orange are translated in bacteria by tRNAs containing cmo5U, those in green by tRNAs with mnm5U, those in blue by tRNAs with mnm5s2U, and those in yellow by tRNAs with cmnm5Um. Codons highlighted in gray are translated by tRNAs with L34 or I34.

CMO5U MODIFICATION

All E. coli tRNAs with a cmo5U modification also have a purine at nucleotide 35 and read codons from fourfold degenerate codon boxes (Fig. 1). The pathway for cmo5U formation is incomplete, as the enzyme responsible for conversion of uridine to 5-hydroxyuridine (ho5U) remains unknown (24). Installation of the carboxylmethyl group is achieved in E. coli by carboxy-S-adenosyl-L-methionine synthase (CmoA), which generates an unusual Cx-SAM moeity, and tRNA U34 carboxymethyltransferase (CmoB), which transfers Cx-SAM to ho5U (Fig. 2) (25–27). The presence of the cmo5U modification was originally proposed on theoretical grounds to expand the base pairing ability of the tRNA beyond Watson-Crick and wobble pairing so as to include the ability to form the cmo5U:U base pair (18). Subsequent in vivo work using knockouts of individual tRNA or modifying enzyme genes demonstrated that some but not all cmo5U34-containing tRNAs are able to efficiently decode even NNC codons (16, 27). For example, Salmonella enterica decodes its four CCN proline codons with three tRNAs, but all four codons can be read with the single tRNAProcmo5UUG species to maintain robust cell growth in the absence of G34- and C34-containing isoacceptors (27). Similarly, E. coli tRNAVal and tRNAAla species with cmo5U34 can partially rescue growth phenotypes arising from knockdown of the other cellular valine and alanine tRNAs (16). However, the expanded decoding efficiency is not used equally, even within a given species. A similar attempt to decode all S. enterica ACN threonine codons with a single tRNAThrcmo5UGU was not successful (16). Depletion of the S. enterica cmoB gene results in tRNAs devoid of cmo5U; inefficient decoding of G-ending proline, valine, and alanine codons demonstrated that cmo5U is important even for the predicted “normal” wobble pairing to occur (16).

FIG 2.

FIG 2

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Biosynthesis of uridine 5-oxyacetic acid (cmo5U). R represents the 5′-phosphoribosyl group of the tRNA backbone. The enzyme responsible for hydroxylation remains unknown. Carboxy-S-adenosyl-Lmethionine (Cx-SAM) is synthesized by carboxy-S-adenosyl-L-methionine synthase (CmoA) and transferred to ho5U by tRNA U34 carboxymethyltransferase (CmoB).

The structural basis for enhanced decoding by cmo5U incorporation was observed in crystal structures of the Thermus thermophilus 30S subunit in complex with GUN-containing mRNA oligonucleotides and a cmo5U tRNAVal anticodon stem-loop (ASLVal) (28). Surprisingly, both the cmo5U:A and cmo5U: G-containing complexes exhibit Watson-Crick geometry; this indicates that the modified uridine adopts the enol tautomer in the cmo5U:G pair. The cmo5U:U and cmo5U:C pairs exhibit a single hydrogen bond in each case (Fig. 3). The nonwobble geometry enables stacking of cmo5U with ASLVal A35 (position 35 is always a purine for cmo5U-containing tRNAs) (28). While earlier NMR studies on nucleotide monophosphates suggested the ribose of cmo5U would adopt a C2’-endo conformation (18), all four crystal structures exhibit a C3′-endo conformation for the modified uridine (28). A parallel solution study of ASLValUAC in the absence and presence of cmo5U revealed that the modification serves to preorder the anticodon loop for codon binding (29).

FIG 3.

FIG 3

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Observed cmo5U pairs in decoding. R represents the 5′-phosphoribosyl group of the RNA backbone. The observed cmo5U G geometry suggests an enol tautomer for the modified base. Figure adapted from Weixlbaumer et al., Nat. Struct. Mol. Biol., 2007, 14, 498–502 (28).

XM5U MODIFICATIONS

This class of modifications in bacteria include mnm5U, cmnm5Um, and the thiolated derivatives mnm5s2U and cmnm5s2U, which are generated by a network of modifying enzymes in multiple steps (Fig. 4) (30–33). These modifications are parallel to the mcm5U and mcm5s2U nucleotides found in eukaryotic tRNAs (2, 32). The bacterial xm5U modifications (where x is a methylamino or carboxymethylamino moiety) are primarily found in tRNAs that decode two-fold degenerate codon boxes, those split between pyrimidine-ending and purine-ending trinucleotides (33). The exceptions are tRNAArgmnm5UCU and tRNAGlymnm5UCC, which read the purine-ending codons in fourfold degenerate boxes. The formation of xm5U modifications is catalyzed by the heterotetrameric MnmEG complex (previously annotated as TrmE and GidA) (33, 34). MnmEG can use either NH3 or glycine as its substrate resulting in the formation of nm5U or cmnm5U, respectively (33). The cmnm5U is then converted to mnm5U through a FAD-and SAM-dependent manner by MnmC1/2; nm5U conversion to mnm5U requires only the SAM-dependent MnmC2 step (35–37). mnm5U can then be further derivatized to mnm5s2U by the IscS-MnmA enzyme complex using cysteine as the sulfur donor (38). Isolation of tRNA harboring either the mnm5U or the s2U partially modified states indicates that the modifications are independent of each other (39, 40). The original cmnm5U can also be further derivatized by either the IscS-MnmA or TrmL to yield cmnm5s2U or cmnm5Um, respectively (38, 41, 42).

FIG 4.

FIG 4

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Synthesis of xm5U modifications. R represents the 5′-phosphoribosyl group of the tRNA backbone. Installation of s2U is a multi-step sulfur trafficking pathway. Figure adapted from Armengod et al., Biochimie, 94, 1510–1520 (33).

MnmE and MnmG are nearly universal in bacteria, missing only in Mycoplasma suis, while TrmL is missing only in six species of mollicutes (43). Conservation of these enzymes suggests that xm5U34 modifications are important for translational fidelity.

Unlike cmo5U, the xm5U modifications do not expand the base pairing potential past the Watson-Crick and wobble pairing seen in unmodified U but they do enhance pairing to codon A and G nucleotides (30–32). An atomic-level perspective was obtained from crystal structures of E. coli tRNALysmnm5s2UUU bound to the T. thermophilus 70S subunit programmed with mRNA containing either AAA or AAG in the A-site (44). The hypermodified wobble base decodes its two cognate codons with distinct geometries (Fig. 5). While the mnm5s2U34-A pair has canonical Watson-Crick pairing, the mnm5s2U34 G did not adopt a typical wobble conformation, in which the U shifts toward the major groove of the codon–anticodon duplex. Instead, the modified uridine shifts toward the minor groove and apparently makes two hydrogen bonds to the N1 and N2 atoms of the codon guanine. The authors rationalize this geometry as arising from either a zwitterionic modified base or an unusual enol tautomer. One possible ionization state and one possible tautomer is shown (Fig. 5).

FIG 5.

FIG 5

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Observed mnm5s2U pairs in decoding. R represents the 5′-phosphoribosyl group of the RNA backbone. The mnm5s2U-A pair exhibits Watson-Crick geometry (top), while the mnm5s2U G base edge distances suggest the modified uridine is either ionized (bottom left) or adopts an enol tautomer (bottom right). Figure adapted from Rozov et al., Nat. Commun., 7, 10457 (44).

ROLE OF S2U MODIFICATION

tRNAs containing U34 together with U35 (those decoding Gln, Glu, and Lys codons) are further modified by thioketone substitution at the O2 position of U34 (45–48). As demonstrated in both solution studies and crystal structures, the larger atomic radius of sulfur compared to oxygen forces a ribose C3′-endo conformation (13). This further enhances stacking interactions with U35, serving to preorder the anticodon for ribosomal decoding. The s2U is thought to restrict and stabilize the conformation of both U34 and U35 in these cases (49, 50). NMR experiments of tRNALys ASL in various states of modification showed that not only does the 2-thio modification promote base stacking between U34 and 35 but also with nucleotide 36, enhancing the interactions between nucleotides 35 and 36 of the anticodon and position one and two of the codon (51, 52).

Early studies using both chemical derivatization and sulfur starvation demonstrated the importance of the s2U modification for aminoacylation of tRNALys, tRNAGln, and tRNAGlu by their corresponding aminoacyl-tRNA synthetases (AARSs) (48, 53). The role of s2U34 in these tRNAs was revealed by comparing in vitro transcribed tRNAs devoid of modifications with partially or fully modified tRNAs. These experiments demonstrated that s2U34 contributed more to aminoacylation activity than did other modifications, including the xm5U34 substitutions (47). In particular the KM of the transcript tRNAGlu was 200-fold higher than that of the native tRNAGlu, suggesting that the wobble modification is a strong identity element for E. coli GluRS (54). The importance of the s2U34 modification in aminoacylation may also be derived from its influence on overall tRNA structure. Among the 20 aminoacyl-tRNA synthetases, only three require tRNA binding for the first step of aminoacylation, amino acid activation by condensation with ATP to form an enzyme-bound aminoacyl-adenylate. These three are the class I enzymes GluRS, GlnRS, and ArgRS (55). Modification-dependent structural variation was observed upon chemical probing of E. coli tRNAGlu “modivariants,” tRNAGluUUC species isolated from cells with modification heterogeneity due to over-production of the tRNA on a plasmid (56). Kinetic parameters for glutamyl-adenylate formation (in the presence of tRNAGlu) and aminoacylation were also impaired by about two orders of magnitude (6). This led to the suggestion that lack of the thioketone in particular may alter the structure of tRNAGlu in a manner that impacts both adenylate formation and amino acid transfer.

Biochemical observations regarding 2-thioketone importance were further validated when crystal structures for GlnRS: tRNAGln and LysRS:tRNALys complexes were solved, as both enzymes make direct contact with the modified wobble position (57, 58). One question that remained was how AARSs can recognize both modified and unmodified anticodon bases in a set of isoaccepting tRNAs. Crystal structures of E. coli GlnRS bound to tRNAGln isoacceptors reveal that the enzyme makes several hydrogen bonds with C34 of tRNAGlnCUG but U34 of the unmodified tRNAGlnUUG isoacceptor lacks any favorable hydrogen bonds (7). Furthermore, the s2U moiety of fully modified tRNAGlncmnm5s2UUG is nestled tightly into a pocket of GlnRS that contains both polar and hydrophobic groups. In the corresponding structure of unmodified tRNAGlnUUG the 2-oxo group is displaced 1.5 Å from the protein compared to the location of the 2-thio group, suggesting that the protein pocket is ideally suited for the large, polarizable sulfur moiety and that unmodified uridine is electrostatically excluded (7). Thus the enzyme is conformationally plastic with respect to its cognate anticodons while binding only weakly to the tRNA with an unmodified U34.

The importance of s2U34 in bacterial tRNAGln, tRNAGlu, and tRNALys isoacceptors is highlighted by the conservation of the thiouridylase enzyme MnmA, which is proposed to be in the minimal set of genes required for bacteria and is retained even in organisms with severely condensed genomes (43, 59). The network of sulfur relay enzymes required to enable tRNA thiolation by MnmA is not conserved, however. Instead of the IscS-MnmA complex described above, Bacillus subtilis and other gram-positive bacteria use an alternate path that includes the cysteine desulfurase YrvO and direct sulfur transfer to MnmA (60).

tRNAs THAT DECODE NNA CODONS WITHOUT U34

The CGA and the AUA codons in E. coli (highlighted in gray, Fig. 1) are translated by tRNAs lacking the expected U34. The CGA arginine codon is translated by a tRNA originally transcribed with an ACG anticodon, which is then modified by a tRNA-specific adenosine deaminase (TadA) enzyme to form the mature inosine base (61, 62). The AUA isoleucine codon is translated by a tRNA originally transcribed with a CAU anticodon, canonically dedicated to reading the lone AUG methionine codon. The specificity of this minor tRNAIle2 is switched from AUG to AUA by incorporation of a lysidine modification (L) at the wobble position (8, 63). These two modification pathways will be discussed separately.

INOSINE MODIFICATION

Inosine at position 34 expands the base pairing potential of a tRNA anticodon to include cytosine, adenosine, and uridine, as predicted in Crick’s original wobble hypothesis (14). Inosine was the first nonstandard nucleotide identified in a tRNA anticodon (64). Deamination of A34 to generate inosine is catalyzed in bacteria by TadA (Fig. 6), likely evolved from a cytosine deaminase (65). tRNAArgACG is the canonical target of TadA, which is essential in E. coli and other bacteria that use inosine to decode the arginine GCU/C/A codons (43, 59). Eukaryotes have a wider range of I34-containing tRNAs, produced by the action of adenosine deaminase acting on tRNA (ADAT) (66); archaea lack I34 tRNAs (67). A recent tRNAome analysis of A34 distribution suggested that some bacteria could expand their use of inosine-wobbling tRNAs. Indeed, Oenococcus oeni tRNALeuAAG was shown to contain I34, presumably generated by TadA (65). Although essential in E. coli, TadA is not widely distributed (4, 43, 68). Loss of TadA function correlates with the emergence of new tRNA genes to cover all arginine codons (69).

FIG 6.

FIG 6

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Inosine formation. R represents the 5′-phosphoribosyl group of the tRNA backbone. A34 is deaminated by bacterial tRNA adenosine deaminase A (TadA) to produce I34-containing tRNAArg.

LYSIDINE MODIFICATION

The AUN codon box (Fig. 1) is the only position in the Universal Genetic Code where purine ending codons (NNR) are split between two amino acids, isoleucine and methionine. In all other cases either both NNR codons are assigned to the same amino acid or one is a stop codon. Use of a tRNAIleUAU to decode the AUA isoleucine codon would be detrimental to translational accuracy because of the potential to decode the AUG methionine by standard wobble. Thus the AUN codon box presents an example of cellular wobble avoidance. To circumvent isoleucine versus methionine ambiguity, each branch in the tree of life has evolved a distinct conserved strategy for AUA decoding. Bacteria use tRNA isoleucine lysidine synthetase (TilS) to convert immature tRNAIle2CAU to tRNAIle2LAU, which enables efficient AUA translation and AUG exclusion (Fig. 7) (63, 70, 71). Archaea adopt a parallel modification strategy in which tRNA isoleucine agmatidine synthetase (TiaS) generates agmatidine on the tRNAIle2CAU wobble position (5, 72, 73). Eukaryotes use the expanded decoding capacity of inosine to translate the three isoleucine codons (74).

FIG 7.

FIG 7

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Lysidine synthesis. Lysidine (k2C or L) enables decoding of the minor AUA isoleucine codon while preventing wobble with the AUG methionine codon.

Lysidine (k2C or L) is formed by the attachment of a lysine moiety to the 2-carbon of C34; this reaction is ATP-dependent and proceeds through a cytosine adenylate intermediate (70, 75). Unlike the previously described modifications occurring in tRNAs decoding the NNA codon, lysidine eliminates the potential for base pairing with any codon other than AUA, so here modification restricts rather than expands decoding potential (8, 63).

While there is no available crystal structure of tRNAIle2LAU decoding, the parallel agmatidine-containing tRNAIle2 from Haloarcula marismortui was solved in complex with T. thermophilus 70S ribosomes and an A-site AUA codon (76). The structure suggests a single H-bond between N1 of the codon adenine and the imine N4 of agm2C34. Additionally, the terminal amine of agmatidine makes a hydrogen bond with the down-stream mRNA backbone (76). Similar contacts can be made in the bacterial lysidine-adenine pair (Fig. 8). Lysidine in the wobble position of tRNAIle2 appears to exclude the AUG methionine codon because of steric clash with the guanine exocyclic amine.

FIG 8.

FIG 8

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Proposed lysidine:adenine base pair. There are several tautomers of lysidine that could form one or two hydrogen bonds with adenine; this pair is suggested by the observed agmatidine:adenine structure in the context of ribosome decoding.

The ability of IleRS to aminoacylate both tRNAIle2LAU and tRNAIleGAU suggests that the synthetase does not use lysidine as a positive determinant for aminoacylation. In the crystal structure of Staphylococcus aureus IleRS in complex with tRNAIleGUA, the anticodon adopts an unusual conformation but there is no direct readout of the wobble base (77). As unmodified tRNAIle2CAU is unable to be aminoacylated by E. coli IleRS, it is more likely that the C34 nucleotide serves as an antideterminant for aminoacylation by IleRS than that L34 is a positive identity element (8). However, tRNAIle2CAU productively interacts with E. coli MetRS, which suggests a critical role for TilS in maintaining translational fidelity (8, 9). The extent to which this interaction occurs seems to be species dependent, as MetRS enzymes from varying organisms have a preference for tRNAMet over unmodified tRNAIle2 ranging from twofold to 2000-fold (78). This suggests that in some organisms, unmodified tRNAIle2 is a viable substrate for MetRS while in others it is not a likely substrate. Furthermore, the relative usage frequency of the isoleucine AUA codon varies across bacterial species. Both TilS efficiency and MetRS selectivity may vary with the cellular need for tRNAIle2LAU, and codon-biased gene expression may be a regulatory strategy used by some organisms.

While TilS is among the most highly conserved modifying enzymes in bacteria (present in 98% of bacteria studied in one analysis) (43, 59, 68), there are alternative routes to AUA decoding. A thermosensitive conditionally lethal mutant of tilS was generated in B. subtilis, and suppressor mutants were obtained. The two suppressor variants that occurred outside the tilS gene were located in genes for tRNAIleGAU, which had the wobble position nucleotide mutated to thymine, resulting in a tRNAIleUAU (79). While these suppressors were able to incorporate isoleucine at AUA codons in the absence of TilS, a low rate of mistranslation (AUA to methionine and AUG to isoleucine) was also observed (79). In contrast to the engineered depletion of TilS function in B. subtilis, Mycoplasma mobile lacks the tilS gene entirely (80). While a tRNAIle2UAU is used to translate the AUA codon, discrimination against AUG is maintained. Although the mechanism to ensure accurate AUA/AUG translation is not fully characterized, it seems the M mobile ribosome has adapted to reject the tRNAIle2UAU/AUG wobble (80).

WOBBLE MODIFICATIONS IN TRANSLATIONAL REGULATION

In more than 50 years since the Wobble Hypothesis was first proposed, much has been learned about the extent of chemical modifications present in tRNAs, the enzymes that catalyze their synthesis, and the structural basis for efficient wobble decoding (or its exclusion). This rich knowledge base now enables the study of dynamic changes in modification patterns during the cellular life cycle (81). Given the role of wobble modifications in recognition of particular codons, it is not surprising that modifications to the wobble uridine described here are among those utilized for codon-biased response to cellular stress (82). For example, hypoxia increases the level of cmo5U modification in Mycobacterium bovis tRNAThrUGU, leading to enhanced translation of codon-biased persistence genes (83). Rare codons that require wobble modifications for decoding, such as the isoleucine AUA, are a prime candidate for modification-regulated proteome adaptation.

ACKNOWLEDGEMENTS

This work was supported by NSF MCB-181831 (RWA) and by a pilot grant from the Wake Forest University Center for Molecular Signaling. EMN was supported by an NIH T32 fellowship (GM095440–06) to Wake Forest School of Medicine.

Abbreviations:

AARS

aminoacyl-tRNA synthetase (individual AARS enzymes are named using the 3-letter abbreviations for their cognate amino acids)

ASL

anticodon stem-loop

I

inosine

L

lysidine

N

any standard ribonucleotide (A, C, G, U)

NMR

nuclear magnetic resonance

Footnotes

The authors declare they have no conflict of interest.

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