The role of tRNA identity elements in aminoacyl-tRNA editing

The rules of the genetic code are implemented by the unique features that define the amino acid identity of each transfer RNA (tRNA). These features, known as “identity elements,” mark tRNAs for recognition by aminoacyl-tRNA synthetases (ARSs), the enzymes responsible for ligating amino acids to tRNAs. While tRNA identity elements enable stringent substrate selectivity of ARSs, these enzymes are prone to errors during amino acid selection, leading to the synthesis of incorrect aminoacyl-tRNAs that jeopardize the fidelity of protein synthesis. Many error-prone ARSs have evolved specialized domains that hydrolyze incorrectly synthesized aminoacyl-tRNAs. These domains, known as editing domains, also exist as free-standing enzymes and, together with ARSs, safeguard protein synthesis fidelity. Here, we discuss how the same identity elements that define tRNA aminoacylation play an integral role in aminoacyl-tRNA editing, synergistically ensuring the correct translation of genetic information into proteins. Moreover, we review the distinct strategies of tRNA selection used by editing enzymes and ARSs to avoid undesired hydrolysis of correctly aminoacylated tRNAs.

Due to their propensity to charge tRNAs with the wrong amino acid, ARSs acquired specialized hydrolytic domains to "edit" their aa-tRNA products.These domains, known as "editing" domains, catalyze the hydrolysis of mischarged tRNAs, ensuring that only correctly aminoacylated tRNAs accumulate in the cell (Figure 1A).In addition to the editing domains embedded in ARSs (known as cis-editing domains), aa-tRNA hydrolysis is catalyzed by standalone deacylases (known as trans-editing domains) (Kuzmishin Nagy et al., 2020;Jani and Pappachan, 2022).Cis-and trans-editing domains act as essential quality control checkpoints to maintain the integrity of the genetic code.The importance of aa-tRNA editing is underscored by the negative phenotypes associated with defects in editing domains (Lee et al., 2006;Nangle et al., 2006;Ling and Soll, 2010;Bullwinkle et al., 2014b;Cvetesic et al., 2014;Liu et al., 2014Liu et al., , 2015;;Lu et al., 2014;Kelly et al., 2019;Lant et al., 2019;Zhang et al., 2021).
In contrast to amino acids, ARSs identify their tRNA substrates through an intricate set of structural and sequence features unique to each tRNA (Schimmel et al., 1993;Giege et al., 1998;Giege and Eriani, 2023).These tRNA features, collectively known as identity elements, promote faithful interactions between tRNAs and ARSs, preventing ARSs from cross-reacting with non-cognate tRNAs.Notably, growing evidence indicates that many editing domains rely on the same tRNA elements to gain aa-tRNA specificity and avoid hydrolysis of correctly aminoacylated tRNAs.This tRNA specificity is crucial to elude unintended energy loss due to the depletion of correctly aminoacylated tRNAs and to maintain adequate aa-tRNA supply for protein synthesis.More importantly, the role of tRNA identity elements in aa-tRNA editing highlights how identity elements secure the accurate translation of the genetic code.

tRNA identities
The elements that define the identity of tRNAs for a particular amino acid primarily reside in the tRNA acceptor stem and the anticodon loop (Figure 1B; Giege et al., 1998;Beuning and Musier-Forsyth, 1999;Giege and Eriani, 2023).Positions 1, 72, and 73 in the acceptor stem, and 35 and 36 in the anticodon are major contributors to tRNA selection.These elements act as an operational code to mark tRNAs for aminoacylation by a specific ARS (Schimmel et al., 1993;Ribas de Pouplana and Schimmel, 2001).Identity elements in the acceptor stem are generally recognized in the aminoacylation site of ARSs, whereas dedicated anticodon binding domains mediate the recognition of tRNA anticodon elements.tRNA identity elements are typically conserved within a single domain of life.However, with few exceptions, they diverge across domains of life (Lin et al., 2019).For example, the operational code for aminoacylation of tRNA Pro diverged during evolution from G72 and A73 in bacteria to C72/A73 and C72/C73 in archaea and eukaryotes, respectively (Liu et al., 1995;Stehlin et al., 1998;Burke et al., 2001).These changes in tRNA Pro were accompanied by changes in the selection mechanism of prolyl-tRNA synthetase (ProRS), preventing cross-reaction between ProRS and tRNA Pro from different domains of life (Stehlin et al., 1998;Burke et al., 2001).Similar changes in the operational code of other tRNAs are known (Giege and Eriani, 2023).

The diversity of editing
Seven ARS families have editing domains to proofread aa-tRNA synthesis, whereas five families and superfamilies of trans-editing domains are currently known (Figure 2; Kuzmishin Nagy et al., 2020;Jani and Pappachan, 2022).In most cases, trans-editing domains are evolutionarily related to the editing domains of ARSs, sharing structural homology and, sometimes, substrate specificity.Trans-and cis-editing domains employ diverse mechanisms of substrate selection, which can involve unique characteristics of the amino acid side chain or tRNA features.Most editing domains use steric exclusion and/or chemical mechanisms to differentiate aminoacyl moieties of aa-tRNAs.Consequently, they tend to display relaxed amino acid specificities.For example, bacterial ProXp-ala, a trans-editing domain, hydrolyzes Ala-and Ser-tRNA with similar efficiency (Danhart et al., 2017).In contrast to their aminoacyl moiety selectivity, both trans-and cis-editing domains, with some exceptions, exhibit more robust tRNA specificities.The tRNA selectivity of editing enzymes can be mediated via direct or indirect interactions.These mechanisms of tRNA recognition are discussed in the following section.

Identity elements in aminoacyl-tRNA editing
Accurate recognition of mischarged tRNAs by editing enzymes is essential to avoid deacylation of correctly aminoacylated tRNAs.Because aa-tRNA synthesis requires an ATP molecule, indiscriminate hydrolysis of correctly charged tRNA by editing enzymes would be energetically costly and could impact cell growth and homeostasis by decreasing the available pool of aa-tRNAs for protein synthesis.As discussed in the following subsections, editing domains have evolved distinct mechanisms of substrate selection that ensure hydrolysis of the incorrect aa-tRNAs.Notably, in many cases, the same tRNA identity elements that define aminoacylation are used to gain specificity during editing (Figure 1C).However, lacking tRNA specificity in other cases may offer a functional advantage in acting on diverse mischarged tRNA substrates emerging from different ARSs.AlaRS erroneously synthesizes Ser-and Gly-tRNA Ala .The appended editing domain of AlaRS is responsible for clearing these mischarged products (Figure 2A; Beebe et al., 2003).The editing domain relies on the almost universally conserved wobble base pair G3:U70 to recognize tRNA Ala (Beebe et al., 2008).G3:U70 is also indispensable for tRNA aminoacylation by AlaRS (Hou and Schimmel, 1988;McClain and Foss, 1988).Thus, a single base pair defines tRNA Ala aminoacylation and editing.How the aa-tRNA Ala substrate is transferred from the aminoacylation site to the editing domain remains unknown.Channeling the aa-tRNA Ala between the two active sites would require substantial structural rearrangement of AlaRS to bring the editing domain closer to the aminoacylation domain and prevent complete dissociation of the tRNA (Naganuma et al., 2014).The C-Ala domain could facilitate the movement of the tRNA between the two domains (Guo et al., 2009).Alternatively, the editing domain could bind the tRNA after being released from the aminoacylation domain.Biochemical and biophysical characterization and structural studies are needed to determine the molecular mechanism of aa-tRNA selection by the editing domain of AlaRS.

ThrRS
Most ThrRSs encode a dedicated editing domain that deacylates Ser-tRNA Thr produced in the aminoacylation domain (Dock-Bregeon et al., 2000;Beebe et al., 2004;Korencic et al., 2004).The editing domain is located at the N-terminus of ThrRS and exhibits evolutionary differences.Eukaryotic and bacterial ThrRS have a structurally similar editing domain known as the N2 (Figure 2A).In contrast, the archaeal ThrRS possesses an editing domain structurally homologous to D-aminoacyl-tRNA deacylases (DTD) (Dwivedi et al., 2005;Hussain et al., 2006).Notably, while the N2 and DTD-like domains effectively hydrolyze Ser-tRNA Thr , they display distinct tRNA selectivity.For example, the N2 editing domain of E. coli ThrRS indiscriminately deacylates bacterial and archaeal Ser-tRNA Thr .In contrast, the DTD-like domain of ThrRS from the archaeon Methanosarcina mazei only hydrolyzes archaeal Ser-tRNA Thr (Beebe et al., 2004).Similarly, the editing domain of Pyrococcus abyssi ThrRS was shown to recognize Ser-tRNA Thr while discriminating against other Ser-tRNA substrates (Novoa et al., 2015).These observations suggest that the tRNA specificity of the archaeal ThrRS editing domain may rely on the identity of position 73 (Beebe et al., 2004;Novoa et al., 2015), a conserved U73 in archaeal tRNA Thr .In contrast, the same position is variable in bacterial and eukaryotic tRNA Thr , consisting of A73 or U73 (Lin et al., 2019).Therefore, the N2 domain may have evolved a relaxed specificity that enables deacylation of tRNA Thr with U73 and A73.This relaxed specificity toward N73 is also observed in the aminoacylation of bacterial and eukaryotic tRNA Thr (Hasegawa et al., 1992;Nameki, 1995).In archaea, the role of N73 in aminoacylation is species-specific, with some species lacking N73 specificity (e.g., Haloferax volcanii) and others (e.g., Aeropyrum pernix) strongly depending on U73 (Ishikura et al., 2000;Nagaoka et al., 2002).Consequently, a weak correlation exists between editing and aminoacylation of tRNA Thr in the context of N73.In contrast to N73, the anticodon bases play a more important and conserved role in tRNA Thr aminoacylation (Giege and Eriani, 2023).Although direct evidence of the importance of the anticodon bases in editing is not available, a model based on E. coli ThrRS suggests that tRNA Thr is held by the ThrRS anticodon binding domain, facilitating the CCA-end repositioning from the aminoacylation site to the editing domain (Dock-Bregeon et al., 2004).Whether the DTD-like editing domain of archaeal ThrRS uses a similar mechanism and how it recognizes the U73 is unknown.

Phenylalanyl-tRNA synthetase (PheRS)
The editing activity of PheRS resides in the B3/B4 domain of the β-subunit of the enzyme's heterodimer (Figure 2A).The B3/B4 domain clears aminoacylation errors involving Tyr and meta-Tyr (Roy et al., 2004;Bullwinkle et al., 2014b).This activity of PheRS is essential for preventing mistranslation of Phe codons and maintaining cellular homeostasis.While a detailed investigation of its tRNA specificity is missing, the activity of the PheRS editing domain is affected by changes in the anticodon, as demonstrated by the lack of deacylation of a tRNA Phe G34A mutant (Ling et al., 2009b).Because G34 is an essential element for aminoacylation (Peterson and Uhlenbeck, 1992;Ling et al., 2009b), this result supports a 3 -end translocation model similar to ThrRS N2 editing, in which the anticodon binding domain provides indirect specificity to the editing by holding the tRNA and enabling the transfer of the 3 -end from the aminoacylation site to the editing site (Roy et al., 2004).Whether elements in the acceptor stem or other tRNA regions are directly recognized by the B3/B4 domain of PheRS requires further investigation.

ProRS
ProRS exists in different structural isoforms.In bacteria, the predominant ProRS isoform encodes an editing domain known as the insertion (INS) domain (Figure 2A).The INS domain catalyzes the deacylation of Ala-tRNA Pro , which is incorrectly synthesized in the aminoacylation domain of ProRS.To avoid deacylation of cognate Ala-tRNA Ala , the INS domain relies on the anticodon binding domain (ABD) of ProRS.The ABD offers specificity by interacting with the unique tRNA Pro anticodon bases G35 and G36 (Das et al., 2014).These bases also serve as identity elements for aminoacylation (Liu et al., 1995;Stehlin et al., 1998).Changes in the identity of these bases prevent the binding of ProRS to the tRNA, impeding tRNA aminoacylation and deacylation.In contrast, mutations in the acceptor stem of tRNA Pro are inconsequential for the catalysis of the INS domain.The role of the anticodon sequence in ProRS editing is further supported by the deacylation of Ala-tRNA Ala mutants with a Pro UGG anticodon (Das et al., 2014).The dependency of the INS domain on the anticodon bases suggests that the ProRS ABD anchors the tRNA, enabling the translocation of the tRNA's 3 -CCA end for editing.However, the molecular basis of this process remains poorly understood.
For editing by ValRS's CP1, A73, A35, and C36 are crucial, while other elements like the U4:A69, the anticodon stem U29:A41 base pair, and the core nucleotide G45 moderately contribute to editing (Tardif and Horowitz, 2002).The ValRS CP1's reliance on the anticodon bases suggests that the ABD facilitates the CCAend translocation between the aminoacylation and editing sites.The ValRS-tRNA complex supports this model (Fukai et al., 2000).Similarly, some overlap between elements for aminoacylation and editing has been established for LeuRS, albeit with antagonistic evidence emerging from two bacterial LeuRS models.For E. coli LeuRS, the interaction between G19 in the D-loop and C56 in the T-loop serves as a critical element for aminoacylation and editing (Du and Wang, 2003).However, LeuRS from Aquifex aeolicus, a deep-branching bacterium, may lack robust tRNA specificity for editing as it effectively edits Thr, Val, and Ile from different tRNA substrates (Zhu et al., 2007).Nonetheless, the anticodon stem-loop may contribute to transferring the tRNA acceptor stem from the aminoacylation to the editing site, as a mutation of A35 in tRNA Leu mildly decreases editing (Yao et al., 2008).Structural evidence of LeuRS suggests that the anticodon binding domain holds the tRNA in place while the CCA-end moves from the aminoacylation state to the CP1 domain (Tukalo et al., 2005;Palencia et al., 2012).However, how changes in the tRNA anticodon influence LeuRS editing activity remains unclear.
Unlike ValRS and LeuRS, IleRS editing requires nucleotides that are different from those needed for aminoacylation.Nucleotides 16, 20, and 21 in the D-loop are the principal features that facilitate editing by E. coli IleRS CP1 (Hale et al., 1997).However, a mutant tRNA Ile G16C/ 20/U21G tRNA Ile is deacylated with similar efficiency as wild-type (Farrow et al., 1999).These discrepancies suggest that D-loop bases influence the transfer of the tRNA but not the chemical step of deacylation (Farrow et al., 1999;Nomanbhoy et al., 1999).Notably, the crystal structure of IleRS bound to the tRNA in an editing conformation did not reveal direct interactions between IleRS and the tRNA D-loop (Silvian et al., 1999).Thus, additional biochemical and structural insights are needed to clarify the tRNA specificity of the IleRS CP1 domain, and how the aa-tRNA Ile traffics between the two IleRS active sites is unknown.This could explain if a direct role of identity elements in editing exists.

Trans-editing domains
In contrast to ARSs, trans-editing domains generally lack dedicated RNA binding domains (Figure 2B).Nonetheless, several of these enzyme families have developed tRNA specificities based on recognizing tRNA acceptor stem elements.This recognition may be mediated in the same catalytic domain. 10.3389/fmicb.2024.1437528

INS superfamily
In addition to the INS domain of ProRS, the INS superfamily groups eight families of trans-editing domains, YbaK, ProXpala, ProXp-x, ProXp-ST1, ProXp-ST2, ProXp-7, ProXp-8, and ProXp-9 (Vargas-Rodriguez and Musier-Forsyth, 2013;Kuzmishin Nagy et al., 2020).Most INS superfamily members are found in bacteria, but each family's phylogenetic distribution pattern is unique.For example, ProXp-ala is found in all domains of life, whereas YbaK is present only in bacteria.Except for the INS domain, INS superfamily members are single-domain proteins.Interestingly, while these enzymes share high structural homologies and active site features, they display a wider range of aa-tRNA specificities catalyzed by several aaRSs.These deacylases also display distinct mechanisms of substrate selection, including tRNA recognition.In the following subsections, each family's activities and tRNA specificities are described, except for ProXp-7, ProXp-8, and ProXp-9, whose functions remain unknown (Kuzmishin Nagy et al., 2020).

ProXp-ala
ProXp-ala shares the same activity with the ProRS INS domain (Ahel et al., 2003;Vargas-Rodriguez and Musier-Forsyth, 2013).However, unlike the INS domain, ProXp-ala has a robust selectivity for tRNA Pro based on the acceptor stem bases N72 and N73, which corresponds to G72 and A73 in bacteria and C72 and C73 in eukaryotes (Vargas-Rodriguez and Musier-Forsyth, 2013;Das et al., 2014;Ma et al., 2023).ProXp-ala's specificity prevents crossreaction with Ala-tRNA Ala .Remarkably, ProXp-ala retained its tRNA Pro specificity during evolution from bacteria to eukaryotes, adapting to changes in the identity of the N72 and N73 bases (Vargas-Rodriguez et al., 2020).ProXp-ala is also found fused to the N-terminus of ProRS (lacking an INS domain) in lower eukaryotes from the Stramenopila, Aveolates, and Rhizaria supergroups and the Leishmania and Trypanosoma genera (Ahel et al., 2003;Vargas-Rodriguez et al., 2020;Parrot et al., 2021).Evidence suggests that the ProRS-fused ProXp-ala can discriminate against Ala-tRNA Ala (Figure 2B; Ahel et al., 2003).In plants, ProXp-ala contains a unique C-terminal domain (CTD) that contributes to the enzyme's tRNA binding affinity (Figure 2B; Byun et al., 2022).However, the mechanism of substrate selection still needs to be determined for the ProXp-ala-ProRS fusion and plant ProXpala.

ProXp-ST1 and ProXp-ST2
ProXp-ST1 and ProXp-ST2 are homologous deacylases that catalyze the hydrolysis of Ser-and Thr-tRNAs (Liu et al., 2015).Both enzymes display broad tRNA specificity, recognizing diverse tRNAs, including tRNA Val , tRNA Ile , tRNA Thr , tRNA Ala , and tRNA Lys , all of which are mischarged with either Ser or Thr by the corresponding ARS (Jakubowski, 2012;Liu et al., 2015).Thus, the broad tRNA specificity of ProXp-ST1 and ProXp-ST2 prevents mistranslation caused by Ser and Thr mischarging.Despite their overlapping substrate specificities, only ProXp-ST2 has developed direct tRNA recognition based on A73.This bias for tRNAs with A73 prevents hydrolysis of Ser-tRNA Ser due to the G73 of tRNA Ser (Liu et al., 2015).ProXp-ST1 is indifferent to the identity of N73, but whether it hydrolyzes Ser-tRNA Ser is unknown.Because tRNA Thr has an A73, ProXp-ST1 and ProXp-ST2 can efficiently hydrolyze Thr-tRNA Thr in vitro.However, ThrRS effectively prevents Thr-tRNA Thr from both enzymes, offering a mechanism that protects correctly aminoacylated tRNA Thr (Liu et al., 2015).A ProXp-ST1-related deacylase, FthB, that hydrolyzes fluorothreonyl-tRNA Thr also exists, but little is known about its tRNA specificity (McMurry and Chang, 2017).

D-aminoacyl-tRNA deacylase (DTD)
DTDs prevent the cellular accumulation of D-aa-tRNAs stemming from several ARSs (Calendar and Berg, 1967;Soutourina et al., 2000).Three distinct DTD isoforms are found in organisms from all domains of life: DTD1 in most bacteria and eukaryotes, DTD2 in plants and archaea, and DTD3 in cyanobacteria (Kumar et al., 2022).Bacterial DTD requires a purine (A/G) in position 73 for effective aa-tRNA deacylation (Kuncha et al., 2018b).The specificity of bacterial DTD enables deacylation of several tRNA substrates while preventing deacylation of Gly-tRNA Gly , which has a conserved U73 in bacteria (Routh et al., 2016).Interestingly, N73 evolved from U to A73 in cytosolic tRNA Gly .This change in the identity of N73 prompted a switch in the tRNA specificity of eukaryotic DTD1, which prefers pyrimidine instead of purine (Gogoi et al., 2022).Whether the identity of N73 plays a role in the deacylation of D-aa-tRNAs is yet to be determined.

Outlook
Despite the strong correlation between the role of identity elements in tRNA editing and aminoacylation, our overall knowledge is limited.The tRNA specificities of several editing enzymes are unknown or poorly understood.For example, whether the B3/B4 domain of PheRS relies on tRNA acceptor stem is still unknown.The lack of molecular tools to prepare aa-tRNA substrates has significantly contributed to our poor understanding of the relationship between identity elements and editing.Producing mischarged tRNA variants using ARSs is challenging because mutating identity elements results in poor aminoacylation.Most available data for the tRNA specificity determination of CP1 domains are based on ATP consumption assays (Farrow et al., 1999;Tardif and Horowitz, 2002;Du and Wang, 2003;Zhu et al., 2007).This method integrates the effect of tRNA mutations in aminoacylation and editing.Thus, establishing the direct contribution of tRNA elements to editing can be intricate because the same elements can impact aminoacylation.The development of flexizyme technology now offers a powerful tool to investigate the role of identity elements in aa-tRNA editing (Murakami et al., 2006).This catalytic RNA ligates virtually any amino acid to tRNAs regardless of their sequence.Thus, it enables the preparation of diverse aa-tRNA mutant substrates to examine identity elements in the context of editing comprehensively (Das et al., 2014;Liu et al., 2015;Novoa et al., 2015;Danhart et al., 2017;Vargas-Rodriguez et al., 2020;Watkins et al., 2024).Adopting flexizyme can help establish and clarify the substrate specificities of many cis-and trans-editing enzymes from diverse species and across domains of life.Ultimately, this will expand our understanding of the dual role of identity elements in editing and aminoacylation, which, in turn, can provide novel insights into the contribution of editing enzymes to the establishment of the genetic code (Beebe et al., 2003).
FIGURE 1 (A) Steps in tRNA aminoacylation and editing.tRNAs are aminoacylated by ARSs producing aa-tRNAs.If the ARS uses a non-cognate amino acid (ncaa), the resulting ncaa-tRNA can be hydrolyzed by the editing enzymes.In the absence of editing checkpoints, the ncaa is incorporated into proteins in response to the wrong codon, causing mistranslation.(B) Representative secondary structures of tRNAs.As discussed in the main text, the numbered bases indicate the various positions important for editing.(C) Summary of the transand cis-editing domains with characterized functions and their known tRNA recognition elements.a Archaeal origin; b indicates weak or no tRNA specificity; c the specificity of N73 depends on the DTD's origin; d in the context of tRNA Ala ; e Bacterial origin; "ND" indicates not determined.B and E for ProXp-Ala indicate bacterial and eukaryotic, respectively.