Evidence for Late Resolution of the AUX Codon Box in Evolution

Background: Protein biosynthesis requires accurate tRNA aminoacylation.

Results: Bacterial methionyl-tRNA synthetases (MRSs) vary in their ability to reject near-cognate tRNA^Ile transcripts containing the methionine-specifying CAU anticodon.

Conclusion: Given the degree of near-cognate discrimination among bacterial MRSs, aspects of genetic code accuracy likely were fixed relatively late in evolution.

Significance: This varied discrimination may reflect differing cellular needs for translational accuracy versus plasticity.

Abstract

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNA^Ile isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNA^Ile precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNA^Ile that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNA^Ile rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNA_CAU^Met from near-cognate tRNA_CAU^Ile. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNA^Ile identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades.

Introduction

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein biosynthesis at the ribosome. Accurate recognition of cognate tRNA by each aminoacyl-tRNA synthetase is essential as tRNAs charged with noncognate amino acids may be used by the ribosome to incorrectly decode codons, introducing mutations into proteins (1). In general, there are 20 aminoacyl-tRNA synthetases, one for each of the standard amino acids (2). (Individual aminoacyl-tRNA synthetases are abbreviated using the standard one-letter amino acid abbreviation.) Methionyl-tRNA synthetase (MRS)⁴ attaches methionine to two distinct tRNA isoaccepting species: initiator tRNA^fMet for decoding the initial AUG (start) codon and elongator tRNA^Met for decoding internal AUG codons, respectively. All tRNA^Met species contain a CAU anticodon. However, the single methionine codon is similar to one of the isoleucine codons (AUA). In fact, these two codons represent the only place in the genetic code where trinucleotides differing only in the type of purine in the wobble (3rd) position specify different amino acids. Solutions to this unique decoding problem vary widely across the tree of life. Higher eukaryotes use a tRNA_UAU^Ile whose U34 is post-transcriptionally modified to pseudouridine, allowing accurate decoding of AUA codons (3). Mitochondria sidestep the recognition problem by using a modified genetic code that assigns both AUA and AUG codons to methionine (4). Bacteria use a tRNA_CAU^Ile whose position 34 cytidine in the anticodon is post-transcriptionally modified by lysidine-tRNA synthase (TilS) to lysidine (L); tRNA_LAU^Ile then specifically decodes the AUA codon (5–7). In archaea, agmatidine is introduced into this same position instead of lysidine (8, 9).

In Escherichia coli, lysidine modification switches the amino acid identity of tRNA from methionine to isoleucine and its decoding capacity from AUG methionine codons to AUA isoleucine codons (5–7). This mechanism is consistent with the prior determination that MRS from E. coli (EcMRS) belongs to an aminoacyl-tRNA synthetase group that uses cognate anticodon nucleotides as dominant identity elements for aminoacylation (10). For such synthetases, the same anticodon residues are responsible both for aminoacylation identity and for decoding at the ribosome; in these enzymes, there is just a single code, the genetic code, which is functioning in translation.

The strength of this anticodon recognition element is illustrated by the determination that EcMRS can methionylate in vitro transcripts of the E. coli tRNA^Ile major isoacceptor in which its GAU anticodon is changed to the methionine CAU (11). However, Mycoplasma penetrans MRS (MpMRS) rejects in vitro transcripts of M. penetrans tRNA_CAU^Ile lacking the lysidine 34 (L34) modification, whereas EcMRS aminoacylates the M. penetrans tRNA_CAU^Ile transcripts just as efficiently as its cognate tRNA^Met transcripts (12). Our results therefore point out a difference in tRNA_CAU^Ile discrimination between the E. coli and M. penetrans methionylation systems. MpMRS utilizes residues in the acceptor stem, in particular an A3-U70 base pair, to discriminate against tRNA_CAU^Ile even without its lysidine modification (12).

Loosely defined, recognition of non-anticodon tRNA residues constitutes an operational code that matches amino acid with tRNA and is distinct from the genetic code (13). As our previous study compared tRNA_CAU^Ile discrimination between just two bacterial MRS enzymes (12), we sought to determine whether strong tRNA_CAU^Ile discrimination and the use of this operational code by MpMRS are the exception or the rule for bacterial MRSs. This question is of particular interest because the operational code dictated by the acceptor stem and discriminator base of the tRNA is proposed to be the ancestral recognition mechanism for early tRNA precursors (13–15).

As the isoleucyl-tRNA synthetase and MRS catalytic sites have evolved in conjunction with tRNA_CAU sequences (16), there appear to have been some sequence motifs preferred by each of the synthetases. Acceptor stem nucleotides are tied with tRNA_CAU^Ile and tRNA_CAU^Met identity in bacteria; G3-C70 and A3-U70 base pairs are both commonly found in tRNA_CAU^Ile, whereas tRNA_CAU^Met and tRNA_CAU^fMet typically have C3-G70 base pairs (17). C4-G69 and C5-G68 also occur frequently in tRNA_CAU^Ile. Although these preferences are likely dictated by subtle constraints inherent in the active site of the two different enzymes, the degree of difference between tRNA_CAU^Ile and tRNA_CAU^Met varies significantly among species. E. coli and other gammaproteobacteria encode a tRNA_CAU^Ile with a C3-G70 base pair common also to its tRNA_CAU^Met and tRNA_CAU^fMet species (17). Thus the key 3-70 base pair used by MpMRS to reject tRNA_CAU^Ile is not used by EcMRS to differentiate its cognate from near-cognate tRNA_CAU (12).

Variation in the zinc binding domain among MRS enzymes might account for tRNA_CAU^Ile discrimination differences as this domain is proposed to play a role in acceptor stem recognition (18, 19). The zinc binding domain forms part of the connective polypeptide linking the halves of the Rossmann fold; depending on the organism, one or two small “knuckle” structures are present (20, 21). Strikingly, MRSs of bacterial origin have only a single knuckle, whereas MRSs of the archaeal clade have two (19, 22). Each knuckle that binds zinc typically consists of four cysteine residues in two CXXC motifs (18), although in some enzymes, the knuckle structure is generated without cysteine residues or coordination of zinc. Thus bacterial MRSs can first be divided as bacterial or archaeal in origin, and then they can be further delineated into two additional classes depending on whether they are predicted to bind one or no zinc ions (in the bacterial clade) or one or two zinc ions (in the archaeal clade) (19, 22).

In this work, we investigate the distribution of tRNA_CAU^Ile discrimination in bacterial methionylation systems. We selected seven systems from different bacterial clades to explore a variety of phylogenetic and structural characteristics. We considered MRS enzymes of eubacterial origin as well as those thought to result from archaeal gene transfer (22). These species furthermore possess a diverse set of tRNA_CAU acceptor stems. The seven enzymes also represent each of the four structural classes of MRS with respect to zinc occupancy in the connective polypeptide domain (19). Finally, the species vary with regard to the type of TilS (tRNA^Ile lysidine synthetase) enzyme they encode: either the longer Type I TilS with an appended tRNA binding domain or the shorter Type II TilS (23, 24). We observe in this set of MRS enzymes a stark variation in levels of tRNA_CAU^Ile discrimination with little correlation to the structural and phylogenetic features described above. Our conclusions support the hypothesis that tRNA_CAU discrimination was established recently with respect to extant bacterial clades.

EXPERIMENTAL PROCEDURES

Materials

DNA oligonucleotides were synthesized by IDT-DNA. l-[³⁵S]Methionine and MonoQ and HisTrap nickel columns were from Amersham Biosciences. Restriction enzymes were from New England Biolabs, and vector pCR2.1-TOPO was from Invitrogen. Expression vector pQE-70 was from Qiagen. PfuUltra DNA polymerase and XL-10 Gold were from Stratagene. M. penetrans genomic DNA was a gift from Dr. Y. Sasaki of the National Institute of Infectious Diseases, Japan. Genomic DNA from Helicobacter pylori strain 26695 (ATCC BAA-255), Streptococcus pneumoniae strain R6 (ATCC BAA-255), Bacteroides fragilis (ATCC 25285), Mycobacterium smegmatis (ATCC 23037), and Borrelia burgdorferi strain B31 (ATCC-35210) were purchased from LGC Promochem.

Transfer RNA Substrate Preparation

Transfer RNA_CAU^Ile and tRNA_CAU^Met sequences were obtained from the Lowe tRNA database (25). RNAs were generated by in vitro transcription of overlapping oligonucleotides (26) using T7 RNA polymerase, 5 mm NTP, 40 mm DTT, 250 mm HEPES·KOH (pH 7.5), 30 mm MgCl₂, 2 mm spermidine, and 0.1 mg/ml bovine serum albumin at 37 °C for 4 h. Transcripts were separated on denaturing PAGE, eluted from the gel using an Elutrap electroelution apparatus (Schleicher & Schuell), and refolded (80 °C followed by gradual reduction of temperature in the presence of 1 mm MgCl₂). Aminoacylation plateaus were used to calculate the concentration of active molecules for each tRNA preparation using 10 μm of the corresponding native enzyme (for tRNA^Met samples) or EcMRS (for tRNA_CAU^Ile samples). The fraction of aminoacylatable tRNA was consistent with E. coli tRNA^Met transcripts we typically use based on A₂₆₀ quantification of tRNA (30–65% active).

Enzyme Cloning

MRS sequences from S. pneumoniae (SpMRS1, GenBank^TM NP_358290.1), H. pylori (HpMRS, GenBank NP_207215.1), B. fragilis (BfMRS, GenBank YP_213853.1), M. smegmatis (MsMRS, GenBank YP_889680.1), and B. burgdorferi (BbMRS, GenBank NP_212721.1) were amplified from genomic DNA and cloned into the pCR2.1-TOPO vector. Each gene was subcloned into the SphI and BamHI sites of the pQE-70 vector (Qiagen) in-frame with a C-terminal His₆ tag. The resulting plasmids pQE-70-SpMRS1, pQE-70-HpMRS, pQE-70-BfMRS, pQE-70-MsMRS, and pQE-70-BbMRS were verified by sequencing. Cloning of MpMRS and EcMRS was previously described (12, 27).

Enzyme Overexpression and Purification

XL-10 Gold cells (Stratagene) transformed with pQE-70-MpMRS, pQE-70-SpMRS1, pQE-70-HpMRS, pQE-70-BfMRS, pQE-70-MsMRS, and pQE-70-BbMRS were grown at 37 °C for 12 h. Protein expression and purification on nickel affinity columns were performed according to the manufacturer's protocol. pQE-70-SpMRS, pQE-70-HpMRS, pQE-70-BfMRS, pQE-70-MsMRS, and pQE-70-BbMRS elutions yielded expected protein bands of 79, 76, 79, 59, and 87 kDa, respectively. pQE-70-MpMRS elution yielded two protein bands of 128 kDa (the full-length protein) and 63 kDa (corresponding to translation from an internal start site that represents the MRS portion of the polypeptide). The 63-kDa MpΔMRS was further purified by ion exchange chromatography as described previously (12). Monomeric E. coli MRS was expressed from a pET28a vector and purified by nickel affinity chromatography as reported (27).

Aminoacylation Assays

Aminoacylation of tRNA was performed at 25 °C in 30-μl reactions containing 20 mm HEPES·KOH (pH 7.6), 100 μm methionine, 10 mm MgCl₂, 5 mm DTT, 4 mm ATP, 150 mm NH₄Cl, 100 μm Na₂EDTA, 0.5 μCi/ml l-[³⁵S]methionine, 4 μm tRNA transcripts, and 50 nm enzyme as reported (28). SpMRS and BbMRS concentrations were later adjusted to 20 and 300 nm, respectively, to ensure linear rates for methionylation of tRNA_CAU^Met and tRNA_CAU^Ile transcripts. BfMRS and MpMRS methionylation of tRNA_CAU^Ile could not be detected at enzyme concentrations low enough to observe linear rates for tRNA_CAU^Met. Thus for these enzymes, methionylation of tRNA_CAU^Met was compared with tRNA_CAU^Ile by using higher concentrations of both enzyme and tRNA_CAU^Ile as described previously (12).

Phylogenetic Analysis

MRS protein sequences were aligned with T-Coffee using standard parameters, and gaps and ambiguously aligned regions were removed (29). Distance trees were calculated with the package PHYLIP, using the programs PROTDIST and NEIGHBOR, and using 1000 bootstraps to estimate robustness of the nodes (30). The overall topology of the tree was confirmed using maximum parsimony using the program PROTPARS in the PHYLIP package and by maximum likelihood using PHYML (31, 32).

RESULTS

Selection of MRS Enzymes

Prior phylogenetic analysis has shown that bacterial MRSs belong to two distinct clades (17). Although many bacterial MRSs appear to be of direct bacterial origin, E. coli and some other species have MRS enzymes apparently resulting from a horizontal gene transfer from archaea. Interestingly, extant archaeal MRS enzymes discriminate against a different C34 modification (agmatidine) than bacterial MRSs (lysidine). On the other hand, eukaryotic cytoplasmic MRSs (arising from archaea) do not require tRNA_CAU^Ile discrimination as eukaryotes do not encode a tRNA_CAU^Ile (25).

We therefore wondered whether the robust ability of EcMRS to aminoacylate tRNA_CAU^Ile (12) might also be observed in other bacterial MRS enzymes of archaeal origin because archaea use a different AUA/AUG decoding strategy. Although previous phylogenetic analyses placed numerous mycoplasma MRSs in the bacterial clade, they did not analyze the position of MpMRS (22). We performed our own phylogenetic analysis of MRS sequences, including those of enzymes assayed in this work. The overall tree architecture was consistent with the previously published work (22). MpMRS was placed in the bacterial clade (Fig. 1), and all the other species tested in this work correctly clustered with species from different families within their same bacterial phylum.

FIGURE 1.

Phylogenetic distance tree of MRS sequences. MRS of archaeal and bacterial origins are depicted in the upper and lower sections, respectively. Species tested in this work are in bold with their corresponding level of tRNA_CAU^Ile discrimination boxed beside them. Bootstrap numbers from 1000 bootstrap replicates are depicted to provide an estimate of the robustness of the nodes. The full sequence alignment is given in supplemental Fig. S1.

We thus selected three MRS examples from the bacterial clade and two from the archaeal clade to expand our initial observations on EcMRS (archaeal clade) and MpMRS (bacterial clade). The selected enzymes also represent the four different zinc binding domain classes (Fig. 2). From the bacterial clade, the examined species include MRS from the proteobacterium H. pylori (HpMRS), the GC-rich M. smegmatis (MsMRS), the firmicute S. pneumoniae (SpMRS1), and the opportunistic pathogen M. penetrans (MpMRS). From the archaeal clade, we selected MRS from E. coli (EcMRS), the spirochete B. burgdorferi (BbMRS), and the obligate anaerobe B. fragilis (BfMRS).

FIGURE 2.

Alignment of bacterial MRS “zinc knuckle” regions. Sequences of MRS representatives used in this work were aligned by CLUSTALW (40); zinc binding motifs are highlighted in yellow, and knuckle pseudomotifs (no bound zinc) are highlighted in gray (19).

The genes for these enzymes were cloned, overexpressed in E. coli, and purified. The corresponding tRNA_CAU^Met and tRNA_CAU^Ile (supplemental Fig. S2) were transcribed, and the transcript quality was confirmed by plateau aminoacylation assays with the MRS of each species for tRNA^Met transcripts and with EcMRS for all tRNA_CAU^Ile transcripts.

Bacterial Clade MRSs Show Either Moderate or Strong tRNA_CAU^Ile Discrimination

Enzymes from bacterial clade species H. pylori, S. pneumoniae, M. smegmatis, and M. penetrans were assayed for their ability to aminoacylate their cognate tRNA_CAU^Met and near-cognate tRNA_CAU^Ile transcripts. The ratio of tRNA_CAU^Met to tRNA_CAU^Ile aminoacylation initial rate was calculated for each MRS as a measure of discrimination efficiency. Enzymes displaying greater than 1000-fold difference in discrimination (as previously seen for MpMRS) were termed strong discriminators, whereas enzymes showing greater than 10-fold but less than 1000-fold difference were termed moderate discriminators. HpMRS, MsMRS, and SpMRS showed moderate discrimination, charging tRNA_CAU^Met more efficiently than tRNA_CAU^Ile by 20-, 40-, and 90-fold, respectively (Table 1). The aminoacylation profile of HpMRS provides an example of the moderately discriminating enzymes with methionylation of tRNA_CAU^Ile observed using nanomolar enzyme concentration. This aminoacylation, whereas modest, is clearly above background and reflects multiple turnover of enzyme (Fig. 3). These results are consistent with tRNA_CAU^Ile discrimination being widely distributed throughout the bacterial clade.

TABLE 1.

tRNA_CAU^Ile discrimination in relation to MRS, tRNA, and cellular properties

SpMRS was used at 20 nm to aminoacylate 2.37 μm tRNA; MsMRS, HpMRS, and EcMRS were each used at 50 nm to aminoacylate 4 μm tRNA; and BbMRS was used at 300 nm to aminoacylate 4 μm tRNA. For inefficiently aminoacylated tRNA^Ile substrates, elevated enzyme and tRNA were used to obtain discrimination values.

Enzyme	Initial rate of tRNACAUMet	Initial rate of tRNACAUIle	Discrimination	MRS clade	Zinc knuckles	Zinc ions	tRNACAUIle 3-70a	tRNACAUMet 3-70a	% of GC genome	TilS typeb
	nm/sec	nm/sec
BfMRS	2.3c	0.46c	2000×	Archaeal	2	1	G-C	C-G	43.1	I
MpMRS	1.5d	1.5d	1600×	Bacterial	1	1	A-U	C-G	25.7	II
SpMRS	6.0	0.069	90×	Bacterial	1	0	A-U	C-G	39.7	I
MsMRS	3.3	0.083	40×	Bacterial	1	0	G-C	C-G	67.4	II
EcMRS	18	0.29	60×	Archaeal	2	1	C-G	C-G	50.8	I
HpMRS	3.3	0.18	20×	Bacterial	1	1	A-U	C-G	38.9	II
BbMRS	3.7	1.9	2×	Archaeal	2	2	G-C	U-A	28.2	I

^a Full tRNA sequences are given in supplemental Figure S2.

^b Type I TilS enzymes possess two tRNA binding domains, whereas Type II TilSs possess one tRNA binding domain.

^c BfMRS was used at 40 nm to aminoacylate 2 μm cognate tRNA_CAU^Met and at 2 μm to aminoacylate 15.5 μm near-cognate tRNA_CAU^Ile.

^d MpMRS was used at 12.5 nm to aminoacylate 2 μm cognate tRNA_CAU^Met and at 1.55 μm to aminoacylate 20 μm near-cognate tRNA_CAU^Ile.

FIGURE 3.

Discrimination by H. pylori MRS. Top, cloverleaf secondary structures of H. pylori tRNA_CAU transcripts. Bottom, discrimination against tRNA_CAU^Ile by H. pylori MRS. Methionylation by 50 nm HpMRS of 4 μm H. pylori tRNA_CAU^Met transcript was 20-fold more efficient than that of 4 μm H. pylori tRNA_CAU^Ile transcript, as determined by comparing initial rates of aminoacylation.

Among the enzymes tested, all bacterial-type MRSs could be classified as discriminating. Representative MRSs from major taxons including firmicutes and proteobacteria were able to reject near-cognate tRNA_CAU^Ile. The extent of discrimination varies widely, however, as the strongly discriminating MpMRS also falls within the bacterial clade.

The zinc binding domain class does not correlate with the level of discrimination. MpMRS and HpMRS both have domains with one knuckle and one zinc ion (Fig. 2), but MpMRS is a strongly discriminating MRS with a 1600-fold difference in charging tRNA_CAU^Met over tRNA_CAU^Ile, whereas HpMRS shows only a moderate 20-fold level of discrimination.

Similarly, although the A3-U70 base pair of M. penetrans tRNA_CAU^Ile is a key identity element for discrimination by MpMRS (12), it is not a predictor of strong discrimination by other enzymes. Like M. penetrans, H. pylori and S. pneumoniae encode tRNA_CAU^Ile with A3-U70 base pairs and tRNA_CAU^Met with C3-G70 base pairs (supplemental Fig. S2), yet their corresponding enzymes exhibit only moderate levels of discrimination (20- and 90-fold, respectively). This result is consistent with the prior observation that EcMRS efficiently aminoacylates M. penetrans tRNA_CAU^Ile transcripts with the A3-U70 base pair (12); thus A3-U70 is not a universal tRNA_CAU^Ile identity element.

Archaeal Clade MRSs Display Great Diversity in Near-cognate Discrimination

The archaeal clade MRSs tested exhibit a striking diversity in their ability to discriminate tRNA_CAU^Ile. Initial assays with BfMRS showed no aminoacylation of tRNA_CAU^Ile at enzyme concentrations that catalyzed efficient aminoacylation of tRNA_CAU^Met. As in previous work with MpMRS (12), we estimated the degree of discrimination using a higher concentration of enzyme and tRNA_CAU^Ile when compared with that used for the tRNA_CAU^Met sample (Fig. 4). Aminoacylation of tRNA_CAU^Ile was observed under these conditions, although multiple turnover of the enzyme was not achieved, and we estimate from initial rates of aminoacylation that BfMRS shows a 2000-fold level of discrimination.

FIGURE 4.

Discrimination by B. fragilis MRS. Top, cloverleaf secondary structures of B. fragilis tRNA_CAU transcripts. Bottom, strong discrimination against tRNA_CAU^Ile by B. fragilis MRS. A 2000-fold difference in catalytic efficiency was estimated by comparing initial rates for methionylation of 2 μm B. fragilis tRNA_CAU^Met by 40, 20, or 10 nm BfMRS to methionylation of 15.5 μm B. fragilis tRNA_CAU^Ile by 2 μm BfMRS.

The strong discrimination observed for BfMRS is even higher than that seen for the bacterial clade MpMRS and clearly distinct from the moderate 60-fold discrimination seen for the archaeal clade EcMRS. This was an unexpected result as BfMRS has the same two knuckle, one zinc ion configuration as EcMRS. Examination of the zinc binding domains does not suggest significant differences in the motifs or flanking sequences (Fig. 2).

Strikingly, the archaeal-type BbMRS exhibited only a 2-fold difference in aminoacylation efficiency between its cognate and near-cognate transcripts (Fig. 5). BbMRS is the only example of a nondiscriminating MRS identified in this work. This observation raises the critical question: How does B. burgdorferi prevent misinsertion of methionine at isoleucine codons?

FIGURE 5.

Discrimination by B. burgdorferi MRS. Top, cloverleaf secondary structures of B. burgdorferi tRNA_CAU transcripts. Bottom, efficient methionylation of tRNA_CAU^Ile by B. burgdorferi MRS. Methionylation by 300 nm BbMRS of 4 μm B. burgdorferi tRNA_CAU^Ile transcript was only 2-fold less efficient than that of 4 μm B. burgdorferi tRNA_CAU^Met transcript, as determined by comparing initial rates of aminoacylation.

Despite the use of the 3-70 base pair as a strong determinant for many MRSs, the tRNA_CAU^Ile and tRNA_CAU^Met of B. burgdorferi contain different 3-70 base pairs. The B. burgdorferi tRNA_CAU^Ile has a G3-C70 base pair versus U3-A70 in tRNA_CAU^Met. Thus BbMRS cannot rely on the 3-70 base pair as an identity element for methionylation.

The B. fragilis tRNA_CAU^Ile has an acceptor stem that is identical to the B. burgdorferi tRNA_CAU^Ile at 12 of 15 nucleotides, including the G3-C70 base pair. However, BfMRS rejects its near-cognate tRNA with 2000-fold discrimination, and BbMRS is nondiscriminating (2-fold aminoacylation difference) despite their common archaeal clade assignment. B. fragilis tRNA_CAU^Ile does have an unusual U6-U67 mismatch in its acceptor stem, but the significance of this pair is unclear. The evolutionary relationship of Bacteroides to other bacteria is somewhat ambiguous, and Bacteroides species comprise their own taxon. BfMRS clusters with the MRS of Cytophaga hutchinsonii and also some archaea such as P. abyssi but is phylogenetically distant from BbMRS and EcMRS and their associated clusters (22) (Fig. 1). It is unclear whether strong tRNA_CAU^Ile discrimination among the MRS of the archaeal clade is restricted to the Bacteroides taxon or whether it is more widely distributed. Clearly, bacterial or archaeal ancestry alone does not determine tRNA_CAU^Ile discrimination ability.

DISCUSSION

The results presented in this work indicate that the extent of tRNA_CAU^Ile discrimination by MRS varies widely in bacteria and is not defined by phylogenetic position or by the structural type of the zinc binding domain. Although we showed earlier that the 3-70 base pair is an important element for MpMRS tRNA_CAU^Ile rejection, its presence or absence is not an accurate predictor of discrimination across bacteria.

Each of the bacterial clade MRSs tested to date aminoacylates its cognate tRNA_CAU^Met at least 20-fold better than its corresponding near-cognate tRNA_CAU^Ile. In contrast, an example of nondiscrimination exists in the archaeal-type BbMRS. BbMRS clusters phylogenetically with eukaryotic MRSs that also have two zinc ions and do not require the same type of near-cognate discrimination as eukaryotes lack tRNA_CAU^Ile. However, a single case of nondiscrimination is not sufficient to define a specific relationship between the zinc binding domain class and discrimination. Additional bacterial enzymes with two zinc ions should be assayed to see whether there is indeed a correlation. Furthermore, characterization of the full set of identity elements used by BbMRS is a priority as its cognate tRNA^Met acceptor stem contains the unusual U3-A70 pair.

We have also examined the genetic background and biochemical environment of each species for putative selective pressures for robust tRNA_CAU^Ile discrimination. The need for acceptor stem-based discrimination in MpMRS could be tied to the high AT bias of the M. penetrans genome, one of the highest of any bacteria (33). The AUA isoleucine codon usage is only 0.4% for the E. coli genome but 2.1% for the M. penetrans genome (34). Increased usage of the AUA codon could potentially make M. penetrans more sensitive to occasional misreading of AUA codons by unmodified Met-tRNA_CAU^Ile, thus driving the need for a stronger MpMRS tRNA_CAU^Ile discrimination mechanism than required by E. coli. However, this hypothesis is not supported by the analysis of the five additional species tested here. The low GC content B. burgdorferi genome uses AUA codons at almost double the frequency of M. penetrans, but BbMRS does not discriminate (Table 1). Likewise, M. smegmatis is a high GC content bacteria with only one AUA codon per 1000, yet MsMRS does exhibit tRNA_CAU^Ile discrimination (34).

A selective pressure for tRNA_CAU^Ile discrimination may stem from the efficiency of the TilS enzyme in each species. MRS enzymes lacking acceptor stem-based discrimination rely on the L34 modification to avoid methionylating tRNA_CAU^Ile. Such enzymes would be expected to methionylate any unmodified tRNA_CAU^Ile present in the cytoplasm. A recent study using a temperature-sensitive Bacillus subtilis TilS did indeed show that loss of TilS activity was correlated with increased mistranslation of AUA codons with methionine, presumably through mismethionylation of unmodified tRNA_CAU^Ile (35). However, if TilS efficiently modifies tRNA_CAU^Ile to tRNA_LAU^Ile, such a scenario would not arise, and acceptor stem-based tRNA_CAU^Ile discrimination would not be needed to prevent mischarging by MRS. Thus the discrimination disparity observed here for different MRSs may reflect in part the efficiency of TilS in each organism.

Recent work has shown that E. coli TilS efficiently processes pre-tRNA_CAU^Ile into tRNA_LAU^Ile, blocking the opportunity for misacylation because pre-tRNA_CAU^Ile lacks a mature 3′ end (24). Although the authors found no detectable level of unmodified E. coli tRNA_CAU^Ile in vivo, it has been previously demonstrated that a single point mutation in E. coli TilS can raise the percentage of unmodified tRNA_CAU^Ile in the cell to 71% (5, 24).

A significant difference in TilS efficiency across species is plausible because there are two distinct types of TilS in bacteria. M. penetrans encodes a Type II TilS that lacks the additional tRNA binding domain found in Type I TilS (e.g. in E. coli TilS) (23, 24). Although the three species tested with the shortened Type II TilS (M. penetrans, M. smegmatis, and H. pylori) have MRS enzymes that discriminate tRNA_CAU^Ile, other results of this work do not show such a clean correlation (Table 1). Specifically, B. fragilis possesses a Type I TilS with the additional domain, but BfMRS strongly discriminates tRNA_CAU^Ile.

Although the CAU anticodon has long been considered the dominant identity element for aminoacylation by MRS, we show that the presence of this single methionine-specifying anticodon is not sufficient to confer aminoacylation in all bacterial enzymes. Of the enzymes tested, only the archaeal clade BbMRS could be classified as nondiscriminating, with only 2-fold discrimination against tRNA_CAU^Ile. Even the well studied EcMRS aminoacylates its cognate tRNA 60-fold more efficiently than its near-cognate tRNA_CAU^Ile.

Certainly, the in vitro conditions used here only begin to test the ability of organisms to prevent misacylation. In addition to bacterial modification of the wobble nucleotide to lysidine by TilS, other modifications not present in the transcripts used here, codon usage, and the relative concentrations of methionine versus isoleucine and tRNA^Met versus tRNA_CAU^Ile are likely to influence translational accuracy at the level of tRNA aminoacylation. Adaptations to the decoding machinery may also enhance accuracy. For example, Mycoplasma mobile is one of several bacteria that lacks TilS; AUA codons are read by tRNA_UAU^Ile, and wobble pairing to Met AUG codons is not observed (36). This suggests that M. mobile ribosomes have adapted to ensure accuracy through a mechanism independent of known anticodon base modifications.

However, despite the numerous means by which genetic fidelity is maintained, translational accuracy may be more plastic than previously thought. Misincorporation of amino acids into the proteome may provide a cellular advantage, particularly during conditions of oxidative or other growth stress (37). In particular, methionine is attached to noncognate tRNAs at about 1% the level of tRNA^Met aminoacylation in human (HeLa) cells, and misacylation increases up to 10-fold upon viral infection or exposure to reactive oxygen species-inducing agents (38). The mediator of this misacylation in human cells is MRS, and a wide range of noncognate tRNAs is mischarged. Similarly, EcMRS exhibits in vitro mischarging of the near-cognate tRNA_CGU^Thr and tRNA_CCU^Arg in an anticodon-dependent fashion; substitutions either to the tRNA_CNU anticodons or to the EcMRS anticodon binding domain decrease misacylation (39).

The rules governing rejection of near-cognate tRNA are clearly not universal and may reflect a relatively late evolution of discrimination strategies. In this regard, several options appear possible. For example, the final assignment of AUG codons to methionine could be, in itself, a late decision in the development of the code. This seems unlikely given the almost universal utilization of AUG as an initiator codon translated by methionine. Alternatively, a shift in the distribution of isoleucine and methionine codons may have taken place later in evolution. In this case, the current situation in mitochondria could represent the ancestral state, which, due to a universal pressure, was independently resolved into the extant structure of the AUX codon box in different clades. This possibility would explain why the strategies to ensure proper tRNA_CAU^Ile discrimination vary so widely among extant species. Alternatively or additionally, the varying degree of cognate versus near-cognate discrimination displayed by bacterial MRSs may indicate species-specific requirements for plasticity of translational fidelity and relative adaptive ability that might be enabled by low level misacylation.