Critical Review

Bacterial Wobble Modi fications of NNA- Emil M. Nilsson Decoding tRNAs Rebecca W. Alexander *

Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina

Abstract

Nucleotides of transfer RNAs (tRNAs) are highly modi fied, particularly (I34 or L34). The structural basis for many N34 modi fications in both at the anticodon. Bacterial tRNAs that read A-ending codons are espe- tRNA aminoacylation and ribosome decoding has been elucidated, and cially notable. The U34 nucleotide canonically present in these tRNAs is evolutionary conservation of modifying enzymes is also becoming modi fied by a wide range of complex chemical constituents. An addi- clearer. Here we present a brief review of the structure, function, and tional two A-ending codons are not read by U34-containing tRNAs but conservation of wobble modi fications in tRNAs that translate A-ending are accommodated by either or at the wobble position codons. © 2019 IUBMB Life, 000(000):1 –9, 2019

tRNA MODIFICATIONS ribosome (3, 5), dictating aminoacyl-tRNA synthetase activity (6 –9), facilitating peptidyl-tRNA translocation (10), and fine-tuning Base pairing rules established in the context of the DNA double ribosomal kinetics (11, 12). P ost-transcriptional modi fications helix generally hold true in RNA, although the greater structural occur throughout tRNA; however, nucleotides in the anticodon and functional diversity of RNA is facilitated by a wide variety of loop are particularly highly decorated. Nucleotides N34, the wob- chemical modi fications. Transfer RNA (tRNA) serves a key func- ble position, and N37, just outside the anticodon, display a larger tion in genetic information transfer, accurately deciphering each diversity of modi fication chemistry than any other tRNA position trinucleotide codon of messenger RNA (mRNA) to produce the (1,4,13).Thisreviewwillconsidermodi fications at the N34 wob- encoded polypeptide product. tRNA is the most chemically modi- ble position in bacterial tRNAs decoding NNA codons. fied RNA species in any organism, with nucleotide modi fications impacting tRNA structure, dynamics, and function. To date over 100 unique RNA modi fications have been identi fied, more than NNA DECODING 90 of which are found in tRNA (1 –3). On average 17% of tRNA tRNAs translating NNA codons are typically encoded with a uri- nucleotides are post-transcriptionally modi fied, more than dine at position 34 (U34), as expected according to Watson-Crick 10 times the frequency observed in larger RNAs such as ribo- rules, and have the intrinsic potential to decode both NNA and somal RNA (rRNA), which is modi fied at 1 –2% (1, 4). Post- NNG codons by classical wobble pairing (14). Such expanded transcriptional modi fications have been shown to in fluence decoding can be advantageous for an organism, as a single tRNA translational accuracy by directly regulating base pairing on the can read both codons. In some cases, however, codon –anticodon wobble pairing could be detrimental to fidelity. In Abbreviations: AARS, aminoacyl-tRNA synthetase (individual AARS either situation organisms have evolved post-transcriptional enzymes are named using the 3-letter abbreviations for their cognate amino modi fication schemes to enforce the correct decoding function of acids); ASL, anticodon stem-loop; I, inosine; L, lysidine; N, any standard ribo- each tRNA (15, 16). The nature of these modi fications varies nucleotide (A, C, G, U); NMR, nuclear magnetic resonance © 2019 International Union of Biochemistry and Molecular Biology among tRNA isoacceptors and across the domains of life, but the Volume 000, Number 000, Pages 1 –9 high frequency of U34 modi fication is conserved. *Address correspondence to: Rebecca W. Alexander, Department of Chem- istry, Wake Forest University, Winston-Salem, NC 27109. E-mail: [email protected] ESCHERICHIA COLI U34 Received 3 June 2019; Accepted 21 June 2019 MODIFICATIONS DOI 10.1002/iub.2120 Published online 00 Month 2019 in Wiley Online Library There are 16 A-ending trinucleotides in the Universal Genetic (wileyonlinelibrary.com) Code, of which two are the ochre (UAA) and opal (UGA) stop

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Gln highlighted in blue, Fig. 1) (21). tRNA UUG when fully modi fied is found with the 5-carboxymethylaminomethyl-2-thiouridine 5 2 Leu (cmnm sU) at position 34 while tRNA UAA presents a 5-carboxymethylaminomethyl-2´-O-methyluridine (cmnm 5Um) (reading codons highlighted in yellow, Fig. 1) (22, 23). Each cat- egory of modi fication 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.

CMO 5U MODIFICATION All E. coli tRNAs with a cmo 5U modi fication also have a purine at nucleotide 35 and read codons from fourfold degenerate codon boxes (Fig. 1). The pathway for cmo 5U formation is incomplete, as the enzyme responsible fo r conversion of to 5-hydroxyuridine (ho 5U) remains unknown (24). Installation of the The universal genetic code. Codons highlighted in FIG 1 carboxylmethyl group is achieved in E. coli by carboxy-S- orange are translated in bacteria by tRNAs containing adenosyl-L- synth ase (CmoA), which generates an cmo 5U, those in green by tRNAs with mnm 5U, those 5 2 unusual Cx-SAM moeity, and tRNA U34 carboxymethyltransferase in blue by tRNAs with mnm sU, and those in yellow 5 by tRNAs with cmnm 5Um. Codons highlighted in (CmoB), which transfers Cx-SAM to ho U(Fig.2)(25 –27). The 5 gray are translated by tRNAs with L34 or I34. presence of the cmo U modi fication 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 codons. Escherichia coli encodes 29 U34-containing tRNAs to ability to form the cmo 5U:U (18). Subsequent in vivo translate 12 sense codons (highlighted in Fig. 1) (17), leaving work using knockouts of individual tRNA or modifying enzyme two A-ending sense codons to be decoded through adaptation of genes demonstrated that some but not all cmo 5U34-containing other tRNAs. All U34-containing E. coli tRNAs are post- tRNAs are able to ef ficiently decode even NNC codons (16, 27). transcriptionally modi fied at the wobble position with a total For example, Salmonella enterica decodes its four CCN proline of five unique species (15). The most frequent modi fication is codons with three tRNAs, but all four codons can be read with the 5 uridine 5-oxyacetic acid (cmo U), which is observed in single tRNA Pro cmo 5UUG species to maintain robust cell growth in Leu Val Ser Thr Ala tRNA UAG , tRNA UAC , tRNA UGA , tRNA UGU , tRNA UGC , the absence of G34- and C34-containing isoacceptors (27). Simi- Pro 5 Val Ala 5 and tRNA UGG (16, 18). Codons read by cmo U-containing larly, E. coli tRNA and tRNA species with cmo U34 can par- tRNAs are highlighted in orange on the Universal Genetic Code tially rescue growth phenotypes arising from knockdown of the Arg Gly table (Fig. 1). Next, tRNA UCG and tRNA UCC both contain a other cellular valine and alan ine tRNAs (16). However, the 5-methylaminomethyluridine (mnm 5U) to translate codons CGA expanded decoding ef ficiency is not used equally, even within a Glu and GGA (highlighted in green in Fig. 1) (19, 20). tRNA UUC given species. A simila r attempt to decode all S. enterica ACN Lys 5 Thr 5 and tRNA UUU also have mnm U34 but are further differenti- threonine codons with a single tRNA cmo UGU was not suc- ated through the addition of a 2-thio group, forming cessful (16). Depletion of the S. enterica cmoB gene results in 5-methylaminomethyl-2-thiouridine (mnm 5s2U) (reading codons tRNAs devoid of cmo 5U; inef ficient decoding of G-ending proline,

Biosynthesis of uridine 5-oxyacetic acid (cmo 5U). R represents the 5 0-phosphoribosyl group of the tRNA backbone. The enzyme FIG 2 responsible for hydroxylation remains unknown. Carboxy-S-adenosyl-Lmethionine (Cx-SAM) is synthesized by carboxy-S- adenosyl-L-methionine synthase (CmoA) and transferred to ho 5U by tRNA U34 carboxymethyltransferase (CmoB).

2 BACTERIAL WOBBLE MODIFICATIONS OF NNA-DECODING TRNAS valine, and alanine codons demonstrated that cmo 5Uisimportant even for the predicted “normal ”wobble pairing to occur (16). The structural basis for enhanced decoding by cmo 5U incorporation was observed in crystal structures of the Thermus thermophilus 30S subunit in complex with GUN-containing mRNA oligonucleotides and a cmo 5U tRNA Val anticodon stem- loop (ASL Val ) (28). Surprisingly, both the cmo 5U:A and cmo 5U: G-containing complexes exhibit Watson-Crick geometry; this indicates that the modi fied uridine adopts the enol tautomer in the cmo 5U:G pair. The cmo 5U:U and cmo 5U:C pairs exhibit a single hydrogen bond in each case (Fig. 3). The nonwobble geometry enables stacking of cmo 5U with ASL Val A35 (position 35 is always a purine for cmo 5U-containing tRNAs) (28). While earlier NMR studies on nucleotide monophosphates suggested the ribose of cmo 5U would adopt a C2 ’-endo conformation (18), all four crystal structures exhibit a C3 0-endo conformation for Val the modi fied uridine (28). A parallel solution study of ASL UAC in the absence and presence of cmo 5U revealed that the modi fi- cation serves to preorder the anticodon loop for codon bind- ing (29).

XM 5U MODIFICATIONS This class of modi fications in bacteria include mnm 5U, cmnm 5Um, and the thiolated derivatives mnm 5s2U and cmnm 5s2U, which are generated by a network of modifying enzymes in multiple steps (Fig. 4) (30 –33). These modi fications are parallel to the mcm 5U and mcm 5s2U nucleotides found in eukaryotic tRNAs (2, 32). The bacterial xm 5U modi fications (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 trinucleotid es (33). The exceptions are Arg 5 Gly 5 tRNA mnm UCU and tRNA mnm UCC, which read the purine-ending codons in fourfold degenerate boxes. The forma- tion of xm 5U modi fications is catalyzed by the heterotetrameric MnmEG complex (previously annotated as TrmE and GidA) (33,

34). MnmEG can use either NH 3 or glycine as its substrate resulting in the formation of nm 5U or cmnm 5U, respectively (33). The cmnm 5U is then converted to mnm 5U through a FAD- and SAM-dependent manner by MnmC1/2; nm 5U conversion to mnm 5U requires only the SAM-dependent MnmC2 step (35 –37). mnm 5U can then be further derivatized to mnm 5s2U by the IscS-MnmA enzyme complex using cysteine as the sulfur donor (38). Isolation of tRNA harboring either the mnm 5U or the s 2U partially modi fied states indicates that the modi fications are independent of each other (39, 40). The original cmnm 5U can also be further derivatized by either the IscS-MnmA or TrmL to 5 yield cmnm 5s2U or cmnm 5Um, respectively (38, 41, 42). Observed cmo U pairs in decoding. R represents the FIG 3 0 MnmE and MnmG are nearly universal in bacteria, missing 5-phosphoribosyl group of the RNA backbone. The observed cmo 5UG geometry suggests an enol tauto- only in Mycoplasma suis , while TrmL is missing only in six spe- mer for the modi fied base. Figure adapted from cies of mollicutes (43). Conservation of these enzymes suggests Weixlbaumer et al., Nat. Struct. Mol. Biol., 2007, that xm 5U34 modi fications are important for translational 14, 498 –502 (28). fidelity.

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5 0 2 FIG 4 Synthesis of xm U modi fications. R represents the 5 -phosphoribosyl group of the tRNA backbone. Installation of s U is a multi- step sulfur traf ficking pathway. Figure adapted from Armengod et al., Biochimie, 94, 1510 –1520 (33).

Unlike cmo 5U, the xm 5U modi fications do not expand the conformation (13). This further enhances stacking interactions base pairing potential past the Watson-Crick and wobble with U35, serving to preorder the anticodon for ribosomal pairing seen in unmodi fied U but they do enhance pairing decoding. The s 2U is thought to restrict and stabilize the confor- to codon A and G nucleotides (30 –32). An atomic-level perspec- mation of both U34 and U35 in these cases (49, 50). NMR tive was obtained from crystal structures of E. coli experiments of tRNA Lys ASL in various states of modi fication Lys tRNA mnm5s2UUU bound to the T. thermophilus 70S subunit showed that not only does the 2-thio modi fication promote base programmed with mRNA containing either AAA or AAG in the stacking between U34 and 35 but also with nucleotide A-site (44). The hypermodi fied wobble base decodes its two cog- 36, enhancing the interactions between nucleotides 35 and nate codons with distinct geometries (Fig. 5). While the 36 of the anticodon and position one and two of the codon mnm 5s2U34-A pair has canonical Watson-Crick pairing, the (51, 52). mnm 5s2U34 G did not adopt a typical wobble conformation, in Early studies using both chemical derivatization and sulfur which the U shifts toward the major groove of the codon – starvation demonstrated the importance of the s 2U modi fication anticodon duplex. Instead, the modi fied uridine shifts toward for aminoacylation of tRNA Lys , tRNA Gln , and tRNA Glu by their the minor groove and apparently makes two hydrogen bonds to corresponding aminoacyl-tRNA synthetases (AARSs) (48, 53). the N1 and N2 atoms of the codon guanine. The authors ratio- The role of s 2U34 in these tRNAs was revealed by comparing nalize this geometry as arising from either a zwitterionic modi- in vitro transcribed tRNAs devoid of modi fications with partially fied base or an unusual enol tautomer. One possible ionization or fully modi fied tRNAs. These experiments demonstrated that state and one possible tautomer is shown (Fig. 5). s2U34 contributed more to aminoacylation activity than did other modi fications, including the xm 5U34 substitutions (47). In Glu particular the K M of the transcript tRNA was 200-fold higher Glu 2 than that of the native tRNA , suggesting that the wobble ROLE OF S U MODIFICATION modi fication is a strong identity element for E. coli GluRS (54). tRNAs containing U34 together with U35 (those decoding Gln, The importance of the s 2U34 modi fication in aminoacylation Glu, and Lys codons) are further modi fied by thioketone substi- may also be derived from its in fluence on overall tRNA struc- tution at the O2 position of U34 (45 –48). As demonstrated in ture. Among the 20 aminoacyl-tRNA synthetases, only three both solution studies and crystal structures, the larger atomic require tRNA binding for the first step of aminoacylation, radius of sulfur compared to oxygen forces a ribose C3 0-endo activation by condensation with ATP to form an

4 BACTERIAL WOBBLE MODIFICATIONS OF NNA-DECODING TRNAS 5 2 0 5 2 FIG 5 Observed mnm sU pairs in decoding. R represents the 5 -phosphoribosyl group of the RNA backbone. The mnm sU-A pair exhibits Watson-Crick geometry (top), while the mnm 5s2UG base edge distances suggest the modi fied uridine is either ionized (bottom left) or adopts an enol tautomer (bottom right). Figure adapted from Rozov et al., Nat. Commun., 7, 10457 (44). enzyme-bound aminoacyl-adenylate. These three are the class I enzyme is conformationally plastic with respect to its cognate enzymes GluRS, GlnRS, and ArgRS (55). Modi fication-dependent anticodons while binding only weakly to the tRNA with an structural variation was observed upon chemical probing of unmodi fied U34. Glu Glu 2 Gln Glu E. coli tRNA “modivariants, ” tRNA UUC species isolated The importance of s U34 in bacterial tRNA , tRNA , and from cells with modi fication heterogeneity due to over- tRNA Lys isoacceptors is highlighted by the conservation of the production of the tRNA on a plasmid (56). Kinetic parameters thiouridylase enzyme MnmA, which is proposed to be in the for glutamyl-adenylate formation (in the presence of tRNA Glu ) minimal set of genes required for bacteria and is retained even and aminoacylation were also impaired by about two orders of in organisms with severely condensed genomes (43, 59). The magnitude (6). This led to the suggestion that lack of the network of sulfur relay enzymes required to enable tRNA thio- thioketone in particular may alter the structure of tRNA Glu in a lation by MnmA is not conserved, however. Instead of the IscS- manner that impacts both adenylate formation and amino acid MnmA complex described above, Bacillus subtilis and other transfer. gram-positive bacteria use an alternate path that includes the Biochemical observations regarding 2-thioketone impor- cysteine desulfurase YrvO and direct sulfur transfer to tance were further validated when crystal structures for GlnRS: MnmA (60). tRNA Gln and LysRS:tRNA Lys complexes were solved, as both enzymes make direct contact with the modi fied wobble position (57, 58). One question that remained was how AARSs can rec- ognize both modi fied and unmodi fied anticodon bases in a set tRNAs THAT DECODE NNA CODONS of isoaccepting tRNAs. Crystal structures of E. coli GlnRS bound to tRNA Gln isoacceptors reveal that the enzyme makes several WITHOUT U34 Gln hydrogen bonds with C34 of tRNA CUG but U34 of the The CGA and the AUA codons in E. coli (highlighted in gray, Gln unmodi fied tRNA UUG isoacceptor lacks any favorable hydro- Fig. 1) are translated by tRNAs lacking the expected U34. The gen bonds (7). Furthermore, the s 2U moiety of fully modi fied CGA codon is translated by a tRNA originally tran- Gln 5 2 tRNA cmnm sUUG is nestled tightly into a pocket of GlnRS scribed with an ACG anticodon, which is then modi fied by a that contains both polar and hydrophobic groups. In the tRNA-speci fic deaminase (TadA) enzyme to form the Gln corresponding structure of unmodi fied tRNA UUG the 2-oxo mature inosine base (61, 62). The AUA codon is group is displaced 1.5 Å from the protein compared to the loca- translated by a tRNA originally transcribed with a CAU antico- tion of the 2-thio group, suggesting that the protein pocket is don, canonically dedicated to reading the lone AUG methionine ideally suited for the large, polarizable sulfur moiety and that codon. The speci ficity of this minor tRNA Ile2 is switched from unmodi fied uridine is electrostatically excluded (7). Thus the AUG to AUA by incorporation of a lysidine modi fication (L) at

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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 Ile amino acid or one is a stop codon. Use of a tRNA UAU to decode the AUA isoleucine codon would be detrimental to translational accuracy because of the potential to decode the AUG methio- nine by standard wobble. Thus the AUN codon box presents an example of cellular wobble avoidance. To circumvent isoleucine Inosine formation. R represents the 5 0-phosphoribosyl FIG 6 versus methionine ambiguity, each branch in the tree of life has group of the tRNA backbone. A34 is deaminated by evolved a distinct conserved strategy for AUA decoding. Bacte- bacterial tRNA adenosine deaminase A (TadA) to pro- Arg ria use tRNA isoleucine lysidine synthetase (TilS) to convert duce I34-containing tRNA . Ile2 Ile2 immature tRNA CAU to tRNA LAU , which enables ef ficient AUA translation and AUG exclusion (Fig. 7) (63, 70, 71). the wobble position (8, 63). These two modi fication pathways adopt a parallel modi fication strategy in which tRNA will be discussed separately. isoleucine synthetase (TiaS) generates agmatidine Ile2 on the tRNA CAU wobble position (5, 72, 73). Eukaryotes use the expanded decoding capacity of inosine to translate the three isoleucine codons (74). INOSINE MODIFICATION Lysidine (k 2C or L) is formed by the attachment of a Inosine at position 34 expands the base pairing potential of a moiety to the 2-carbon of C34; this reaction is ATP-dependent tRNA anticodon to include cytosine, adenosine, and uridine, as and proceeds through a cytosine adenylate intermediate (70, 75). predicted in Crick ’s original wobble hypothesis (14). Inosine was Unlike the previously described modi fications occurring in tRNAs the first nonstandard nucleotide identi fied in a tRNA anticodon decoding the NNA codon, lysidine eliminates the potential for (64). Deamination of A34 to generate inosine is catalyzed in bac- base pairing with any codon other than AUA, so here modi fica- teria by TadA (Fig. 6), likely evolved from a cytosine deaminase tion restricts rather than expands decoding potential (8, 63). Arg Ile2 (65). tRNA ACG is the canonical target of TadA, which is essen- While there is no available crystal structure of tRNA LAU tial in E. coli and other bacteria that use inosine to decode the decoding, the parallel agmatidine-containing tRNA Ile2 from Halo- arginine GCU/C/A codons (43, 59). Eukaryotes have a wider arcula marismortui was solved in complex with T. thermophilus range of I34-containing tRNAs, produced by the action of adeno- 70S ribosomes and an A-site AUA codon (76). The structure sine deaminase acting on tRNA (ADAT) (66); archaea lack I34 suggests a single H-bond between N1 of the codon adenine tRNAs (67). A recent tRNAome analysis of A34 distribution and the imine N4 of agm 2C34. Additionally, the terminal suggested that some bacteria could expand their use of inosine- amine of agmatidine makes a h ydrogen bond with the down- Leu wobbling tRNAs. Indeed, Oenococcus oeni tRNA AAG was shown stream mRNA backbone (76). Similar contacts can be made in to contain I34, presumably generated by TadA (65). Although the bacterial lysidine-adenine pair (Fig. 8). Lysidine in the essential in E. coli , TadA is not widely distributed (4, 43, 68). Loss wobble position of tRNA Ile2 appears to exclude the AUG of TadA function correlates with the emergence of new tRNA methionine codon because of st eric clash with the guanine genes to cover all arginine codons (69). exocyclic amine.

2 FIG 7 Lysidine synthesis. Lysidine (k C or L) enables decoding of the minor AUA isoleucine codon while preventing wobble with the AUG methionine codon.

6 BACTERIAL WOBBLE MODIFICATIONS OF NNA-DECODING TRNAS Ile the tilS gene were located in genes for tRNA GAU , which had the wobble position nucleotide mutated to thymine, resulting in Ile a tRNA UAU (79). While these suppressors were able to incor- porate isoleucine at AUA codons in the absence of TilS, a low rate of mistranslation (AUA to methionine and AUG to isoleu- cine) was also observed (79). In contrast to the engineered depletion of TilS function in B. subtilis ,Mycoplasma mobile Ile2 lacks the tilS gene entirely (80). While a tRNA UAU 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 Ile2 M. mobile ribosome has adapted to reject the tRNA UAU /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 Proposed lysidine:adenine base pair. There are sev- FIG 8 modi fications present in tRNAs, the enzymes that catalyze their eral tautomers of lysidine that could form one or two hydrogen bonds with adenine; this pair is suggested synthesis, and the structural basis for ef ficient wobble decoding by the observed agmatidine:adenine structure in the (or its exclusion). This rich knowledge base now enables the context of ribosome decoding. study of dynamic changes in modi fication patterns during the cellular life cycle (81). Given the role of wobble modi fications in recognition of particular codons, it is not surprising that modi fi- Ile2 The ability of IleRS to aminoacylate both tRNA LAU and cations to the wobble uridine described here are among those Ile tRNA GAU suggests that the synthetase does not use lysidine as utilized for codon-biased response to cellular stress (82). For a positive determinant for aminoacylation. In the crystal struc- example, hypoxia increases the level of cmo 5U modi fication in Thr ture of Staphylococcus aureus IleRS in complex with Mycobacterium bovis tRNA UGU , leading to enhanced transla- Ile tRNA GUA , the anticodon adopts an unusual conformation but tion of codon-biased persistence genes (83). Rare codons that there is no direct readout of the wobble base (77). As require wobble modi fications for decoding, such as the isoleu- Ile2 unmodi fied tRNA CAU is unable to be aminoacylated by E. coli cine AUA, are a prime candidate for modi fication-regulated IleRS, it is more likely that the C34 nucleotide serves as an proteome adaptation. antideterminant for aminoacylation by IleRS than that L34 is a Ile2 positive identity element (8). However, tRNA CAU productively interacts with E. coli MetRS, which suggests a critical role for TilS in maintaining translational fidelity (8, 9). The extent to ACKNOWLEDGEMENTS which this interaction occurs seems to be species dependent, as This work was supported by NSF MCB-181831 (RWA) and by a MetRS enzymes from varying organisms have a preference for pilot grant from the Wake Forest University Center for Molecu- tRNA Met over unmodi fied tRNA Ile2 ranging from twofold to lar Signaling. EMN was supported by an NIH T32 fellowship 2000-fold (78). This suggests that in some organisms, (GM095440-06) to Wake Forest School of Medicine. The authors unmodi fied tRNA Ile2 is a viable substrate for MetRS while in declare they have no con flict of interest. others it is not a likely substrate. Furthermore, the relative usage frequency of the isoleucine AUA codon varies across bac- terial species. Both TilS ef ficiency and MetRS selectivity may Ile2 REFERENCES vary with the cellular need for tRNA LAU , and codon-biased [1] Jackman, J. E., and Alfonzo, J. D. (2013) Transfer RNA modi fications: nature ’s gene expression may be a regulatory strategy used by some combinatorial chemistry playground. Wiley Interdiscip Rev RNA 4, 35 –48. organisms. [2] Rozenski, J., Crain, P. F., and McCloskey, J. A. (1999) The RNA modi fication While TilS is among the most highly conserved modifying database: 1999 update. Nucleic Acids Res. 27, 196 –197. enzymes in bacteria (present in 98% of bacteria studied in one [3] Agris, P. F., Narendran, A., Sarachan, K., Väre, V. Y. P., and Eruysal, E. (2017) analysis) (43, 59, 68), there are alternative routes to AUA The importance of being modi fied: the role of RNA modi fications in transla- tional fidelity. Enzyme 41, 1 –50. decoding. A thermosensitive conditionally lethal mutant of tilS [4] Machnicka, M. A., Olchowik, A., Grosjean, H., and Bujnicki, J. M. (2014) Distri- was generated in B. subtilis , and suppressor mutants were bution and frequencies of post-transcriptional modi fications in tRNAs. RNA obtained. The two suppressor variants that occurred outside Biol. 11, 1619 –1629.

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