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. 2024 Mar 1;33(3):e4844. doi: 10.1002/pro.4844

Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase

Claudia Alvarez‐Carreño 1,2,✉, Marcelino Arciniega 3, Lluís Ribas de Pouplana 4,5, Anton S Petrov 1,2, Adriana Hernández‐González 3, Jorge‐Uriel Dimas‐Torres 3, Marco Igor Valencia‐Sánchez 3,6,✉, Loren Dean Williams 1,2,✉, Alfredo Torres‐Larios 3
PMCID: PMC10895455  PMID: 38009704

Abstract

Aminoacyl‐tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA‐modifying proteins. Component domains of bacGlyRS show common ancestry with (i) the catalytic domain of class II tRNA synthetases; (ii) the HD domain of the bacterial RNase Y; (iii) the body and tail domains of the archaeal CCA‐adding enzyme; (iv) the anti‐codon binding domain of the arginyl tRNA synthetase; and (v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain has been found thus far only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined results suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

Keywords: genetic code, GlyRS, translation, tRNA

1. INTRODUCTION

Translation of mRNAs into coded polypeptides is universal to life (Bowman et al., 2020). mRNA decoding during translation depends on the ribosome and a group of enzymes called aminoacyl tRNA synthetases (aaRSs) (Burbaum & Schimmel, 1991; Giegé & Eriani, 2023; Gomez & Ibba, 2020; Woese et al., 2000). aaRSs determine the genetic code by recognizing specific tRNAs and linking them to their cognate amino acids. aaRSs fall into two classes with independent origins in the deep evolutionary past (Eriani et al., 1990; Gomez & Ibba, 2020; Moras, 1992). The catalytic domain of Class I aaRSs is a Rossmann fold while the catalytic domain of Class II aaRSs is an α/β three‐layered sandwich. The early evolution of aaRSs has been linked to the origins of the genetic code and to the emergence of the first proteins (Fournier et al., 2011; Hartman, 1995; Koonin & Novozhilov, 2017; Ribas de Pouplana, 2020; Ribas de Pouplana & Schimmel, 2001).

Throughout the phylogenetic tree, a given tRNA is typically aminoacylated by a single type of aaRS. However, there are exceptions to this rule. There are two distinct lysyl‐tRNA synthetases (LysRSs) (Terada et al., 2002). A class II LysRS is more frequent in bacteria, while a class I LysRS is found predominantly in archaea (Tumbula et al., 1999).

There are also two distinct GlyRSs (Han et al., 2023; Ostrem & Berg, 1974; Tang & Huang, 2005). The GlyRS found in archaea and eukarya, and in some bacteria, is called here arcGlyRS. The GlyRS found in most in bacteria is called here bacGlyRS. Except for their catalytic domains, bacGlyRS and arcGlyRS are globally different (Logan et al., 1995) and use different strategies to recognize tRNAGly. arcGlyRS is relatively small, with a catalytic domain and a tRNA recognition domain. bacGlyRS is larger and more complex. Here we ask why there are two GlyRSs. How did they originate? Which GlyRS appeared first? Why is the distribution of the two GlyRSs over the phylogenetic tree patchy?

We and others (Dimas‐Torres et al., 2021; Han et al., 2023) have hypothesized that the component domains of bacGlyRS were serially exapted through bricolage. The bricolage hypothesis suggests that complex proteins can arise and evolve through stepwise exaptation and adaption of pre‐existing domains or modules, which are combined to produce new functions (Jacob, 1977). In this hypothesis, domains can be thought of as evolutionary building blocks that provide information on ancestry.

If the bricolage hypothesis is correct, homologies of individual domains can be used to help reconstruct the complex histories of multidomain proteins such as bacGlyRS and can help us understand ancestral relationships. Accurate histories require accurate domain delineations. A domain is an quasi‐independent and stable three‐dimensional structure (Levitt & Chothia, 1976; Porter & Rose, 2012; Rossman & Liljas, 1974) and is a common unit of evolution (Ohno, 2013; Ponting et al., 2000). Domains are encoded by genes. Duplicated genes, called paralogs (Fitch, 1970), can combine to form a variety of distinct multidomain proteins.

bacGlyRS contains α and β subunits. The combined data are interpreted here to indicate that the β subunit of bacGlyRS holds six component domains rather than five domains as proposed previously (Ju et al., 2021). The domain of bacGlyRS that escaped detection in previous work is informative about bacGlyRS ancestry and shows homology to a domain found in the universal alanyl tRNA synthetase (uniAlaRS), a class II aaRS. Sequence and structural comparisons suggest that bacGlyRS is more closely related to uniAlaRS than to arcGlyRS (Dimas‐Torres et al., 2021; Smith & Hartman, 2015; Valencia‐Sánchez et al., 2016).

Our results are consistent with a model in which bacGlyRS evolved by bricolage. We show that six component domains of bacGlyRS are homologous to other proteins. These other proteins are universal, bacteria‐specific or archaea‐specific. bacGlyRS shows common ancestry with six domains of four proteins. Each of these proteins interacts with an RNA although none interact with tRNAGly. Component domains of bacGlyRS show sequence and structural similarities with (1) the catalytic and ATL domains (see below) of uniAlaRS; (2) the anti‐codon binding domain (ABD) of the universal arginyl‐tRNA synthetase (uniArgRS); (3) the body and tail domains of the archaeal CCA‐adding enzyme; and (4) the HD domain of the bacterial RNase Y (in agreement with previous observations of structural similarity; Ju et al., 2021). This conservation of RNA as substrate throughout a series of exaptation steps suggests an accessible mechanism by which RNA‐associated proteins are repurposed for new RNA‐associated functions.

2. RESULTS

2.1. bacGlyRS has a complex evolutionary history

The phylogenetic distribution of bacGlyRS appears irregular and discontinuous (‘patchy’) over the extant twigs of the bacterial Tree of Life. We examined a broad range of archaeal and bacterial proteomes, some of which are only partial (Figure 1). Bacterial species have either bacGlyRS or arcGlyRS. Bacteria containing both bacGlyRS and arcGlyRS are extremely rare. Of the 542 sampled proteomes, arcGlyRS (GenBank: PSL06957.1), and bacGlyRS (GenBank: PSL02860.1) are found together only once, in the bacterium Haloactinopolyspora alba (NCBI RefSeq assembly: GCA_003014555.1) isolated from sediment of the Dead Sea. Many bacterial phyla have an irregular distribution of the GlyRSs with multiple branches containing either bacGlyRS or arcGlyRS. Firmicutes, Actinobacteria, Chloroflexi, Proteobacteria as well as DST, PVC, and FCB groups each contain species with bacGlyRS and species with arcGlyRS. Candidate Phyla Radiation (CPR) uniformly contains arcGlyRS; Cyanobacteria uniformly contains bacGlyRS.

FIGURE 1.

FIGURE 1

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Phylogenetic distribution of the two versions of Glycyl tRNA synthetase in Bacteria. The presence of bacGlyRS and arcGlyRS is mapped into a bacterial phylogeny. Yellow dotted lines: bacGlyRS with linked α and β subunits form homodimers instead of heterotetramers. Pink dotted lines: species in which the genes for the α and β subunits overlap. Red dotted lines: species in which the genes for the α and β subunits are separated by nine base pairs or less. Purple dotted lines: species in which the genes for the α and β subunits are separated by more than nine base pairs. The topology of the tree was obtained from (Moody et al., 2022). bp, base pairs; CPR: Candidatus Phyla Radiation; DST, Deinococcus‐Thermus, Synergistetes, Thermotogae, Caldiserica, Coprothermobacterota; F. Fusobacteria; PVC: Planctomycetes, Verrucomicrobia, Chlamydiae, Kiritimatiellaeota, Lentisphaerae, Candidatus Desantisbacteria, Candidatus Omnitrophica; FCB: Fibrobacteres, Chlorobi, Bacteroidetes, Gemmatimonadetes, Candidatus Cloacimonetes, Candidatus Fermentibacteria, Candidatus Glassbacteria; PANNAM, Proteobacteria, Aquificae, Nitrospinae, Nitrospirae, Acidobacteria, Chrysiogenetes, Deferribacteres, Schekmanbacteria and Thermodesulfobacteria.

2.2. bacGlyRS sequences are present in early diverging bacteria

The phylogenetic distribution of bacGlyRS suggests that it was present at the Last Bacterial Common Ancestor (LBCA). bacGlyRS is found in early diverging bacterial phyla (Moody et al., 2022) such as Firmicutes, Acidobacteria, and Proteobacteria (Figure 1). bacGlyRS is not found in the CPR phylum. The presence of arcGlyRS in CPR proteomes may be due to horizontal gene transfer rather than vertical inheritance. The CPR phylum contains putative symbiotic and parasitic bacteria with reduced genomes, large gene losses and extensive lateral gene transfer (Achsel et al., 2001; Jaffe et al., 2020).

2.3. Domains in bacGlyRS are related to RNA‐modifying enzymes

Each of the six domains of bacGlyRS have an independent evolutionary history. Each domain of bacGlyRS displays strong sequence similarity to a domain of another protein (Figure 2). We determined proteins that are the closest sequence matches to each of the six domains of bacGlyRS (Figure 2e). The catalytic domain shows sequence similarity to the catalytic domain of uniAlaRS (e‐value 1.1 × 10−07). The body and tail domains show sequence similarity to domains of the archaeal CCA‐adding enzyme (e‐value 1.4 × 10−08). The HD domain shows sequence similarity to a domain of ribonuclease Y (RNase Y) (e‐value 5.7 × 10−08). The ABD shows sequence similarity to a domain of uniArgRS (e‐value 2.9 × 10−17).

FIGURE 2.

FIGURE 2

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Domain organization of bacGlyRS. Structure of bacGlyRS of Thermoanaerotrix daxensis with naturally linked α and β‐subunits (PDB: 7LU4). (a) Surface representation of bacGlyRS indicating the region that corresponds to the α‐(light purple) and β‐(light blue) subunits in other bacteria. (b) A ribbon representation of bacGlyRS shows structural units (domains) in different colors: dark blue, catalytic domain; light purple, unknown domain 1; dark orange, body domain; light orange, tail domain; light blue, unknown domain 2; yellow, HD domain; green, ATL domain; and dark purple, ABD domain. (c) Domains and structural units of bacGlyRS. (d) Residue range of the domain annotations for E. coli (UniProt IDs: P00960 and P00961) and T. daxensis (UniProt ID: A0A0P6Y0P9). (e) Proteins containing domains with high‐scoring sequence similarity by bidirectional BLAST.

We identified a previously unrecognized domain in bacGlyRS that we call ancient tRNA latch (ATL) domain. Our determination that ATL is a distinct domain follows previous identification of this domain in uniAlaRS (Naganuma et al., 2009). The ATL domains of uniAlaRS and bacGlyRS are similar to each other in sequence and structure (Figure 3a). If ATL is a distinct domain in uniAlaRS, then it is also a domain in bacGlyRS. Previously, the ATL domain was described incorrectly as a dependent element of the HD domain (Han et al., 2023; Ju et al., 2021; Yu et al., 2023); we infer here from sequence and structural considerations that the ATL is independent of other elements of bacGlyRS. The ATL domain appears to be unique to bacGlyRS and uniAlaRS, no other homologs of the ATL domain are identified in a comprehensive protein sequence database (UniRef90). The ATL domain of uniAlaRS contains a C‐terminal α‐helical decoration that is absent from the ATL domain of bacGlyRS.

FIGURE 3.

FIGURE 3

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Structures of bacGlyRS and uniAlaRS showing regions of homology. (a) Comparison of the relative position of the catalytic and ATL domains of bacGlyRS (PDB: 7YSE) and uniAlaRS (PDB: 3WQY) with respect to their cognate tRNA molecules. Insets: Structure superimposition of the catalytic and ATL domains of bacGlyRS and uniAlaRS. (b) bacGlyRS of Escherichia coli in complex with tRNA. (c) uniAlaRS of Archaeoglobus fulgidus in complex with tRNA. (d) Sequence alignment and indexing of the ATL domains of uniAlaRS and bacGlyRS. (e) Hydrogen bonds between the ATL domain of bacGlyRS and its cognate tRNA. (f) Hydrogen bonds between the ATL domain of uniAlaRS and its cognate tRNA.

In addition, we observe that the function of the ATL domain has been mischaracterized. The ATL domain in uniAlaRS is called the “putative anticodon‐binding domain” in some classification databases (SCOPe ID a.203.1.1; Chandonia et al., 2017, and ECOD ID 613.1.1.1; Schaeffer et al., 2017). However, neither the ATL domain of uniAlaRS nor the ATL domain of bacGlyRS contacts the anti‐codon stem‐loop of tRNA (Kumar et al., 2019; Naganuma et al., 2014). Both interact extensively with the amino acid acceptor stem. This mischaracterization of the ATL domain structure and function has obscured its evolutionary origins and molecular function.

2.4. bacGlyRS and uniAlaRS homology is observed in key functional domains

The primary functions of aaRSs are to recognize tRNAs and amino acids and to covalently join them together. The catalytic and ATL domains, which carry out these essential functions are homologous between bacGlyRS and uniAlaRS.

The ATL domains of uniAlaRS and bacGlyRS recognize tRNAs by variations of a common mechanism. tRNAs are recognized by uniAlaRS and bacGlyRS by interactions of ATL with the tRNA acceptor stem (Han et al., 2023; Ju et al., 2021; Yu et al., 2023). The tRNA acceptor stem is the first seven base pairs of tRNA, the unpaired nucleotide at position 73, and the 3′‐CCA tail. In species containing bacGlyRS, the acceptor stems of tRNAGly and tRNAAla differ in three pairs and in position 73. These three base pairs and position 73 are signatures, meaning that they are highly conserved within each group (Penev et al., 2021), tRNAGly or tRNAAla, but differ between the two groups. The specific signatures of tRNAGly are base pairs C2:G71, G3:C70, G4:C69, and U73. The signatures of tRNAAla are G2:C71, G3:U70, C4:G69, and A73. In uniAlaRS, the wobble base pair G3:U70 is the main identity determinant for tRNAAla aminoacylation by uniAlaRS (Chong et al., 2018; McClain et al., 1991; Naganuma et al., 2009, 2014; Nagato et al., 2023; Yu et al., 2023). The ATL domain interacts with the base of the discriminator position (U73 in tRNAGly, and A73 in tRNAAla).

The ATL domain has adapted to distinguish features of the amino acid acceptor stems of tRNAGly or tRNAAla (Figure 3). We have identified four distinct ATL adaptations, allowing a common fold to recognize different tRNAs. To discriminate between the two different tRNAs, the ATL domain (i) slightly shifts global position relative to the tRNA, (ii) shifts local position of the secondary structural elements by conformational change, (iii) changes sequence, and (iv) acquires α‐helical decorations.

The ATL domains of bacGlyRS and uniAlaRS exhibit slightly different positions relative to the tRNA. A distance of 4.1 Å between the centers of mass of bacGlyATL and AlaATL after direct superimpositions of the tRNAGly in complex with bacGlyRS (PDB 7YSE) and the tRNAAla in complex with uniAlaRS (PDB 3WQY), indicates the changes in the global pose of the ATL domains (Figure 3a).

The interactions between ATL and tRNA are locally different in bacGlyATL and AlaATL. After direct superimposition of the bacGlyATL and AlaATL protein domains, a 3.1 Å root mean square deviation between the superimposed backbone atoms quantitates local changes in conformation (Figure 3a). To describe local positional shifts, we have reindexed the ATL residues so that a common index (subscripted here) indicates the same column in the sequence alignment (Figure 3d). bacGlyATL10 indicates the amino acid at the 10th alignment position of ATL domain of bacGlyRS while AlaATL10 indicates the amino acid of the ATL domain of uniAlaRS at the same alignment position. Differences in local positioning of amino acids within ATL are seen by differences in ATL indexes of a common interaction. For example, local positional shifts are seen in the interaction between bacGlyATL29 and AlaATL28 with the G1 base and the discriminator positions of the tRNAs (U73 in tRNAGly and A73 in tRNAAla) and in the interaction between bacGlyATL35 and AlaATL34 with C72 and the discriminator. Base pair G1:C72 is conserved in both tRNAGly and tRNAAla. By contrast, there is no shift at index 26. Both bacGlyATL26 and AlaATL26 interact with position 70, which is a C in tRNAGly and a U in tRNAAla.

The bacGlyATL interaction with tRNA appears intrinsically less stringent than that of AlaATL. The bacGlyATL domain contacts nucleotides of only one strand in the region of C68, C69, and C70. It does not contact the opposing strand (G3, G4, or G5, Figure 3e). By contrast, AlaATL targets both strands of tRNAAla, interacting with both bases of base‐pairs G3:U70 and C4:G69 in part by using the AlaATL‐specific C‐terminal decoration (Figure 3f).

The catalytic domain undergoes a radical change in global position between bacGlyRS and uniAlaRS. In uniAlaRS, the catalytic and ATL domains are directly adjacent in sequence and structure. By contrast, in bacGlyRS, the HD and body‐tail domains are inserted between the catalytic and ATL domains. The HD domain shifts and rotates the catalytic domain of bacGlyRS by 130° compared to the catalytic domain of uniAlaRS (Figure 3b). To accommodate the change in the position of the catalytic domain of bacGlyRS, the tRNA distorts. The conformation of the four unpaired nucleotides of the acceptor arm of tRNAGly bends (Figure 3a), such that the position of the terminal adenosine is shifted by ~26 Å in the bacGlyRS complex compared to in the uniAlaRS complex.

The conformation of the CCA tail and the position of the terminal adenosine appear to be unique in bacGlyRS compared to other aaRSs. The CCA tail of tRNAGly bends away from the anti‐codon in the complex with bacGlyRS (Yu et al., 2023), shifting the terminal A towards the minor groove of the tRNA. All other class II aaRSs bend the CCA tail in the opposite direction, toward the anticodon (Figure S1) or, in the case of the uniAlaRS‐tRNA complex (Figure 3a,c), maintain the relaxed linear structure (Mohan et al., 2009). The position of the terminal adenosine is shifted by ~30 Å in the tRNA complex of bacGlyRS compared to in the PheRS complex (Figure S1).

2.5. The catalytic and ATL domains of bacGlyRS resemble archaeal uniAlaRS homologs

We formulated a tentative model in which bacGlyRS originated in bacteria via duplication of a two‐domain uniAlaRS module. One domain is ATL and the other is the catalytic domain. This tentative model predicts that bacGlyRS component domains would be most similar to bacterial uniAlaRS homologs. Therefore, we investigated the similarity of bacGlyRS domains to archaeal and bacterial uniAlaRS sequences. As shown here this model is not supported by the data.

The sequences of the catalytic and ATL domains in bacterial uniAlaRS and archaeal uniAlaRS are distinctive. A composite multiple‐sequence alignment (MSA) containing both bacterial and archaeal uniAlaRS sequences reveals lineage‐specific signature positions and insertions (Figure S2). Signature positions correspond to columns in a composite MSA that are conserved within groups but are different between groups (Penev et al., 2021). Thus, differences between bacterial and archaeal uniAlaRS sequences reflect deep divergence between bacteria and archaea and are not due to a simple bias in amino acid usage. This type of pattern in a composite MSA is called a block structure and is observed in many universal aaRS and other proteins of the translation system (Vishwanath et al., 2004; Woese et al., 2000).

The catalytic and ATL domains of bacGlyRS resemble archaeal uniAlaRS homologs more closely than bacterial uniAlaRS homologs (Figure 4a,b). We compared the sequences of the catalytic and ATL domains of bacGlyRS with those of uniAlaRS using the program CLANS (Altschul et al., 1990; Frickey & Lupas, 2004) and their profiles using the program HH‐align (Steinegger et al., 2019). Profiles are calculated from MSAs and reflect position‐specific sequence variability. A cluster map of sequences from the catalytic domains of bacGlyRS and uniAlaRS at a p‐value lower than 10−12 shows connections between bacGlyRSs and archaeal uniAlaRSs. At this p‐value threshold, the cluster map lacks connections between bacGlyRSs and bacterial uniAlaRSs (Figure 4a). Similarly, a cluster map of sequences from the ATL domains of bacGlyRS and uniAlaRS at a p‐value threshold of 10−11 shows connections between bacGlyRSs and archaeal uniAlaRSs. This cluster map lacks connections between bacGlyRSs and bacterial uniAlaRSs (Figure 4b). Thus, the catalytic and ATL domains of bacGlyRS are more similar to archaeal homologs than to bacterial homologs. Therefore, our tentative model in which bacGlyRS originated by duplication of a bacterial uniAlaRS module composed of catalytic and ATL domains (Figure 5d) is not supported by the data (Figure 4).

FIGURE 4.

FIGURE 4

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Sequence analysis of bacGlyRS protein domain homologs. Cluster map based on pairwise sequence similarities. Nodes represent sequences; connections between nodes indicate matches between pairs of sequences with a p‐value lower than the threshold. (a) Pairwise sequence similarity between the catalytic domains of bacGlyRS and uniAlaRS. (b) Pairwise sequence similarity between the ATL domains of bacGlyRS and uniAlaRS. (c) Maximum‐likelihood (LG + R4) unrooted tree of catalytic and ATL domains including bacGlyRS and uniAlaRS sequences. small‐AlaRS are sequences from an anomalous subgroup of uniAaRS found in some archaea and bacteria.

FIGURE 5.

FIGURE 5

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Emergence of bacGlyRS through molecular bricolage. (a) Comparative diagram of the multidomain arrangements of bacGlyRS and uniAlaRS. (b) The most probable model. showing emergence of the common ancestor of bacGlyRS and uniAlaRS before the divergence of LUCA. Duplication of the common ancestor gives rise to the proto‐uniAlaRS and proto‐bacGlyRS. The ancestor of bacGlyRS is lost in archaea. (c) Emergence and duplication of the catalytic and ATL domains at LUCA. Before the divergence between archaea and bacteria, one copy of the catalytic domain and one copy of the ATL domain form the uniAlaRS core, the paralogs of the catalytic and ATL domains continue as single‐domain proteins. Secondary loss of the copy of the catalytic and ATL domains in archaea. Formation of the bacGlyRS core in bacteria. (d, e) Evolution of the core of uniAlaRS before the divergence of LUCA. (d) The catalytic and ATL domains are horizontally transferred from the archaeal to the bacterial lineage and formation of the bacGlyRS core. (e) Duplication of the catalytic and ATL domains from uniAlaRS in bacteria and formation of the bacGlyRS core.

2.6. The catalytic and ATL domains represent a functional evolutionary core

Canonical uniAlaRS has five component domains; (i) the catalytic domain, which aminoacylates the tRNA, (ii) the ATL domain, which recognizes tRNAAla, (iii) a cradle loop barrel domain, (iv) an editing domain and (v) a C‐terminal domain (C‐Ala) that binds to the tRNA elbow formed by the D‐ and T‐ loops (Naganuma et al., 2009; Swairjo et al., 2004) (Figures 3c and S1). We identified a subgroup of bacterial and archaeal uniAlaRS sequences that have only three of these domains: catalytic, ATL, and editing (Figures 4 and S1). uniAlaRSs in this subgroup lack the cradle loop barrel domain and the C‐Ala domain of canonical uniAlaRSs (Figure S2). We call this anomalous subgroup small‐AlaRS. We identified small‐AlaRS in the proteomes of the CPR bacterium Candidatus Thermoplasmatota, and in DPANN, and Asgard groups. Small‐AlaRS proteins have bacteria‐specific insertions, suggesting that sequences were transferred to some archaea via horizontal gene transfer. The maximum likelihood tree of catalytic and ATL domain sequences supports a horizontal transfer hypothesis of small‐AlaRS to some archaea (Figures 4c and S3). Small‐AlaRS sequences and recently discovered viral mini‐AlaRS sequences (Antika et al., 2023), which lack the cradle loop barrel, the editing and the C‐Ala domains of canonical cellular uniAlaRSs, support the hypothesis that a core composed of the catalytic and ATL domains can be functional.

The combined data support a model in which bacGlyRS and uniAlaRS emerged before LUCA from common catalytic and ATL ancestors (Figure 5a,b). These catalytic and ATL ancestors were single‐domains and were independent of each other. Other possibilities put the emergence of the bacGlyRS core after the divergence of LUCA. This late emergence of bacGlyRS appears less parsimonious because it suggests that some bacteria lost the ancestral GlyRS and replaced it with an RS protein that has no homologs in other bacteria. Possible scenarios are (1) formation of the proto‐bacGlyRS in the bacterial lineage independently from the uniAlaRS catalytic‐ATL core (Figure 5c); (2) formation of the proto‐bacGlyRS by partial or complete duplication of the uniAlaRS gene in the archaeal lineage, followed by a lateral gene transfer of the proto‐bacGlyRS from archaea to the bacterial lineage; and (3) formation of the proto‐bacGlyRS by partial or complete duplication of the uniAlaRS gene in the bacterial lineage (Figure 5d). This last scenario is not supported by the data (Figure 4).

3. DISCUSSION

bacGlyRS has a deep and convoluted evolutionary history. We infer exaptation relationships (Frenkel‐Pinter et al., 2022) between six bacGlyRS domains and six domains of four different RNA‐modifying proteins (Figure 2). Exaptation is the co‐opting of an existing molecule, domain, trait or system for new function. In our model, upon or after fusion of the ATL and catalytic domains to form ancestors of both bacGlyRS and uniAlaRS, the catalytic domain was exapted to link glycine and tRNAGly to form glycyl‐tRNAGly. The ATL domain was exapted to recognize the acceptor‐stem of the tRNAGly (Han et al., 2023). The prior function of the catalytic domain may have been non‐specific linkage of amino acids to RNAs. The prior function of ALT is unknown. After these processes, insertion of the HD, body and tail domains impacted the relative positions of the ALT and catalytic domains of bacGlyRS and the interaction of the catalytic domain with the CCA tail of tRNAGly (see below). The HD domain was exapted to interact with the discriminator position of tRNAGly (Han et al., 2023). The body and tail domains of bacGlyRS were exapted to interact with the arm and elbow of tRNAGly (Yu et al., 2023). In the archaeal CCA‐adding enzyme, the body and tail domains also interact with the acceptor stem of the tRNA (Schimmel & Yang, 2004; Tomita et al., 2004). The ABD domain, which is homologous to a domain in the class I uniArgRS, and shares ancestry with it, was exapted to interact with the anti‐codon loop of tRNAGly (Yu et al., 2023).

Here, we provide evidence that the ATL domain is a distinct, independent domain of bacGlyRS rather than an integral component of the HD domain as previously proposed. This distinction is important for understanding the deep evolutionary history of aaRSs. Homology between the catalytic and ATL domains of bacGlyRS and the corresponding domains of uniAlaRS suggests common ancestry of the core domains and functions of these enzymes. It is likely that an ancestral class II catalytic domain and an ATL domain joined, and the resulting two‐domain aaRS gave rise to two separate enzymes, one for glycine and another for alanine. This simple model is consistent with sequence and structural similarities between the core domains of bacGlyRS and uniAlaRS.

The conservation of the pose of the ATL domain in uniAlaRS and bacGlyRS, but not the pose of the catalytic domain, is consistent with a model in which the ATL domain is ancestral to both uniAlaRS and bacGlyRS (Figure 5b). It appears that the ATL domain played a role in primitive RNA biology before it was incorporated into these two synthetases. We have identified four adaptations of ATL that allowed a common fold in a common pose to recognize different tRNAs. The ATL domain (i) slightly shifts global position relative to the acceptor stem of the tRNA, (ii) shifts local positions of amino acid backbone and sidechains by conformation change, (iii) changes sequence, and (iv) acquires a C‐terminal decoration in the uniAlaRS that is absent from the bacGlyRS.

bacGlyRS and uniAlaRS use different strategies to achieve final recognition of their cognate tRNAs (Figure 3). ATL domains of both enzymes have similar interactions with the discriminator base and the G1:C72 base pair which is conserved in tRNAGly and tRNAAla. uniAlaRS uses the ATL domain to interact intensively with the acceptor stem, employing the C‐terminal decoration to assist in targeting both strands of the acceptor step. The ATL domain in bacGlyRS lacks C‐terminal decoration and interacts less intensively with the acceptor stem. In the bacGlyRS complex, the ATL domain only contacts nucleotides of one strand of the acceptor stem. To compensate, bacGlyRS interacts with other regions of the tRNA. bacGlyRS but not uniAlaRS exapted additional recognition domains through a process of bricolage.

The conformation of the CCA tail and the position of the terminal adenosine appear to be unique in bacGlyRS compared to other aaRSs. In uniAlaRS, the catalytic and ATL domains are directly adjacent to each other in sequence and structure. tRNAAla interacts with the CCA tail of uniAlaRS in a relaxed state, which is reasonably similar to the structure in the free tRNA (Westhof et al., 1988). In bacGlyRS, insertion of the HD domain caused a translation and rotation of the catalytic domain relative to the ATL domain (Figure 3b). To accommodate this translation/rotation, the acceptor‐arm of tRNAGly in the bacGlyRS complex bends acutely in the direction of the minor groove (Figure 3c). This conformation of the tRNA appears to be unique to bacGlyRS.

The sequences of bacGlyRS and archaeal uniAlaRS share distinctive features. The catalytic and ATL domains of bacGlyRS more closely resemble uniAlaRS archaeal homologs than bacterial homologs (Figure 4a,b). Thus, a model in which bacGlyRS originated within the bacterial lineage by duplication of a bacterial catalytic‐ATL uniAlaRS module is not supported by the data (Figure 5d). The combined results suggest that bacGlyRS and uniAlaRS share a common ancestor composed of the catalytic and ATL domains (Figure 5a). The catalytic and ATL domains represent an evolutionary and functional core. The domains in the shared common core of bacGlyRS and uniAlaRS interact with the most ancient region of the tRNA (Schimmel et al., 1993; Schimmel & de Pouplana, 1995).

Here, we propose a simple model, in which insertion of the body‐tail and HD domains (or possibly just the HD domain) into a gene closely related to the proto‐AlaRS formed a novel enzyme, a proto‐bacGlyRS (Figure 5a). In this model, the interaction between ATL domain and the tRNA remained relatively unchanged as the catalytic domain was displaced and the CCA tail bends towards the minor groove. The proto‐bacGlyRS enzyme underwent adaptations that conferred a new amino acid specificity (Gly) and a new tRNA identity (tRNAGly). It is possible that the new tRNA was better suited to the CCA tail orientation imposed by the HD insertion.

bacGlyRS is one of the largest aaRSs and is usually composed of two subunits, with a catalytic domain in the α subunit and several recognition domains in the β subunit. In most bacteria (Figure 1) bacGlyRS is a heterotetramer (Gomez & Ibba, 2020). In some bacteria, including Thermoanaerotrix daxensis, the α and β subunits are fused as a single polypeptide chain (Figure 1); bacGlyRS with linked α and β subunits form homodimers (Figure 2a). The form of oligomerization, the presence of α‐β linked bacGlyRSs and the encoding of α and β bacGlyRS subunits as a single (Keng et al., 1982; Wagar et al., 1995), and sometimes overlapping (Figure 1 and Table S1), open reading frame, could indicate that the ancestor of all bacGlyRSs was a single α‐β protein that formed homodimers. Fragmentation of bacGlyRS may have occurred later (Wagar et al., 1995) and persists in most species due to evolutionary pressures (Ju et al., 2021). Secondary fragmentation of uniAlaRS has also been reported (Arutaki et al., 2020).

The distribution of bacGlyRS in a wide range of deeply rooted bacterial species suggests origins before LBCA. The presence of bacGlyRS in Terrabacteria and Gracilicutes, the two major bacterial groups (Figure 1) also supports this early origin. However, in the bacterial tree the distribution of bacGlyRS and arcGlyRS appears patchy and irregular. The dominance of arcGlyRS in CPR proteomes suggests that symbiotic bacteria with reduced genomes prefer a smaller enzyme. Currently, we do not have a model that fully explains the discontinuous distribution over evolutionary history.

Our observations here reflect deep evolutionary history. Glycine and alanine appear to be ancient amino acids, both prebiotically available and biologically important. They are among the most abundant amino acids produced in spark discharge experiments (Parker et al., 2011) and are found in chondrite asteroids (Botta et al., 2002). We propose that bacGlyRS and uniAlaRS emerged from a shared core before LUCA (Figure 5a,b), from catalytic and ATL domains that had separate and distinct origins. These prebiotic amino acids share the first and third codon positions, and the proteins that aminoacylate tRNAGly and tRNAAla share sequence and structure relationships. It appears we are seeing evidence of an ancestral protein with non‐specific catalytic activity that may have been able to link either glycine or alanine to an RNA, presumably an ancestor of tRNA. The deep relationship between alanine and glycine is seen in their shared chemistry and biology.

3.1. Methods

3.1.1. Sequence similarity search

A MSAs of bacGlyRS sequences was calculated for a representative set of archaea and bacteria. The MSA was trimmed to the domain boundaries as defined by the structure analysis. The MSAs of the domains were converted to profile Hidden Markov Models using the HHsuite version 3.3.0 (Steinegger et al., 2019). A profile–profile similarity search on the Evolutionary Classification of Domains (ECOD) database was performed with HHsearch using the profiles of bacGlyRS domains as queries. Target domains in ECOD yielding HHsearch probabilities greater than 60% over more than 50 aligned columns were retrieved to serve as queries of a second search on a sequence database containing the proteomes of the representative set of bacterial and archaeal species. The second search was performed using jackhmmer from the HMMER suite version 3.3.1 (Söding, 2005), with default parameters (five iterations). The ECOD database only contains domains that are directly annotated on proteins with experimentally determined structures. The second search aims to identify sequence similarity in the UniProt Reference Cluster, UniRef90 (Lussi et al., 2023); this database includes proteins without experimentally determined structures. Sequences passing the threshold values (E‐value <1 × 10−3, query coverage 80%) were retrieved and trimmed to the region of sequence similarity.

We broadly describe the phylogenetic distribution of the best hits according to the number of sampled proteomes containing homologous sequences as follows: universal (presence in >50% of archaea and > 50% of bacteria); archaea‐specific (presence in >50% of archaea and < 10% of bacteria); bacteria‐specific (presence in <10% of archaea and < 50% of bacteria); lineage‐specific within archaea (presence in <50% of archaea and < 50% of bacteria); lineage‐specific within bacteria (presence in <10% of archaea and < 50% of bacteria sampled); or of complex distribution (presence in 10%–50% of archaea and 10%–50% of bacteria).

3.1.2. Cluster analysis

The query MSAs and trimmed target sequences retrieved from the second sequence similarity search were clustered with CLANS (Frickey & Lupas, 2004) based on pairwise sequence similarities. To find the best sequence match of each query domain, the cut‐offs of the clustering were adjusted to find the p‐value for which the group of query sequences shows sequence relationships with only one other group of orthologs. The subset of best matches was further clustered to reveal possible differences between archaeal and bacterial sequences. Bacterial and archaeal uniAlaRS sequences displayed differential similarity to bacGlyRS sequences on the cluster analysis. To corroborate the clustering analysis results, additional analyses were performed at the level of profiles.

3.1.3. Pairwise profile analysis: HHalign

An MSA containing orthologous sequences of uniAlaRS was further divided to include only archaeal sequences or only bacterial sequences. The MSAs of archaeal and bacterial sequences of uniAlaRS and the MSA of bacGlyRS were trimmed to the catalytic and ATL domains. The MSA of the catalytic domain of bacGlyRS was aligned to the MSAs of the catalytic domains of archaeal and bacterial uniAlaRS sequences using HHalign. The HHalign scores of these MSA‐MSA comparisons agree with the differential pairwise similarities observed in CLANS between archaeal and bacterial sequences.

3.1.4. ML tree

The composite MSA of the catalytic domain containing sequences from bacGlyRS and archaeal sequences from uniAlaRS was concatenated to the composite MSA of the ATL domain. The concatenated MSA was trimmed with trimAl (Capella‐Gutiérrez et al., 2009). A Maximum Likelihood tree was calculated using the trimmed MSA using PhyML (Guindon et al., 2010) and SMS model selection tool (Lefort et al., 2017) on the Montpellier Bioinformatics Platform. The tree was inferred using an LG + R4 model of evolution. Branch support corresponds to a Bayesian‐like transformation of aLRT (aBayes) (Anisimova et al., 2011).

AUTHOR CONTRIBUTIONS

Claudia Alvarez‐Carreño: Conceptualization; writing – original draft; methodology; visualization; writing – review and editing; formal analysis; investigation. Marcelino Arciniega: Writing – original draft; formal analysis; investigation. Lluís Ribas de Pouplana: Writing – review and editing. Anton S. Petrov: Writing – original draft; writing – review and editing; formal analysis. Adriana Hernández‐González: Formal analysis; writing – review and editing. Jorge‐Uriel Dimas‐Torres crystallized bacGlyRS and solved the structure. Marco Igor Valencia‐Sánchez: Writing – review and editing; formal analysis; conceptualization; investigation. Loren Dean Williams: Writing – review and editing; writing – original draft; formal analysis; investigation. Alfredo Torres‐Larios: Conceptualization.

Supporting information

Data S1. Supporting Information.

PRO-33-e4844-s001.pdf (3.8MB, pdf)

ACKNOWLEDGMENTS

The authors dedicate this work to the memory of Professor ATL who passed away on September 9, 2021. Professor ATL worked on the structural determination of the bacterial glycyl tRNA synthetase and performed the initial structural analysis. The authors thank Jessica Bowman, Eric Smith and Arturo Becerra for discussions. This work was funded by the National Aeronautics and Space Administration grant 80NSSC18K1139. CAC was sponsored by the National Aeronautics and Space Administration (NASA) through a contract with ORAU.

Alvarez‐Carreño C, Arciniega M, Ribas de Pouplana L, Petrov AS, Hernández‐González A, Dimas‐Torres J‐U, et al. Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase. Protein Science. 2024;33(3):e4844. 10.1002/pro.4844

Review Editor: Nir Ben‐Tal

Contributor Information

Claudia Alvarez‐Carreño, Email: ccarreno6@gatech.edu.

Marco Igor Valencia‐Sánchez, Email: igorval@comunidad.unam.mx.

Loren Dean Williams, Email: loren.williams@chemistry.gatech.edu.

DATA AVAILABILITY STATEMENT

Sequence alignments and CLANS input files have been deposited in the FigShare repository DOI: 10.6084/m9.figshare.23269007.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Supporting Information.

PRO-33-e4844-s001.pdf (3.8MB, pdf)

Data Availability Statement

Sequence alignments and CLANS input files have been deposited in the FigShare repository DOI: 10.6084/m9.figshare.23269007.

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