Recognition by Tryptophanyl-tRNA Synthetases of Discriminator Base on tRNA Trp from Three Biological Domains*

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA Trp discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis , Archeoglobus fulgidus , and bovine tRNA Trp , representing the three biological domains. When B. subtilis , A. fulgidus , and human TrpRS were used to acylate these tRNA Trp , two distinct preference profiles regarding the discriminator base of different tRNA Trp substrates were found: G > A > U > C for B. subtilis TrpRS, and A > C > U > G for A. fulgidus and human TrpRS. The preference for G73 in tRNA Trp by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Tryptophanylation Tryptophanylation with B. subtilis Tryptophanylation Tryptophanylation M 3 time points, aliquot of the reaction mixture was removed and spotted onto a 3MM filter. Filters acid followed by 95% ethanol wash before to liquid scintillation subtraction of con- trol radioactivity from reaction mixture added tRNA Trp . When the steady-state kinetic parameters k cat and K m for tRNA Trp were to be determined, a saturating concentration of 10 (cid:2) M L -[5- 3 H]tryptophan (10 Ci/mmol, Amersham Pharmacia) for TrpRS was present in the assay mixture. The tRNA concentration was varied over a 10-fold range, all at least 100-fold in excess over TrpRS. Assays were performed in dupli- cates using six tRNA concentrations and two TrpRS concentrations, and errors on individual kinetic constants were consistently less than 15%. Kinetic parameters from three replicate experiments were estimated by fitting data into the Eadie-Hofstee plot using data analysis software Origin 6.1 (Originlab) and averaged.

Aminoacyl-tRNA synthetases catalyze the covalent attachment of amino acids to their cognate tRNA, thereby ensuring the faithful translation of the genetic code (1,2). Cross-species aminoacylation provided a useful phylogenetic probe that generated early evidence for the close relationship between archaea and eukarya (3). The specific recognition of a tRNA by its cognate aminoacyl-tRNA synthetase is guided by identity elements on the tRNA. The discriminator base (N73) has been shown to be a major identity element on tRNA Trp for bacterial and eukaryotic TrpRS 1 (4 -6) as well other tRNAs (7). N73 is strongly conserved in tRNA Trp among different species from the same biological domain, always being G in bacterial and chloroplast tRNA Trp but A in archaeal and most eukaryotic tRNA Trp (8). The reliance on N73 by TrpRS in its specific recognition of tRNA Trp has resulted in one of the first observations of an evolutionary change in identity elements, with bacterial and eukaryotic TrpRS utilizing dissimilar identity elements on the tRNA Trp (4).
To gain further insight into this evolutionary change in N73 recognition, in this study the interactions between TrpRS and tRNA Trp from all three biological domains of bacteria, archaea, and eukarya were compared. Archeoglobus fulgidus is a hyperthermophilic, sulfate-reducing, and strictly anaerobic archaeon, for which the complete genome has been sequenced (9). A complete set of Bacillus subtilis, A. fulgidus, and bovine tRNA Trp carrying different base substitutions at N73 was hyperexpressed in Escherichia coli and examined in terms of their efficiency as substrates for tryptophanylation by B. subtilis, A. fulgidus, and human TrpRS. Moreover, to determine any structural role of N73 in bringing about tryptophanylation, solution NMR spectroscopy was employed to compare the structures of wild-type A73 bovine tRNA Trp and its G73 mutant, which were respectively, the best and the poorest substrates among the bovine tRNA Trp N73 variants toward reaction with human TrpRS. Trp -Three pairs of complementary oligodeoxyribonucleotides separately encoding a T7 promoter sequence upstream of B. subtilis, A. fulgidus, and bovine tRNA Trp genes carrying random nucleotides at position N73 were synthesized according to the gene sequences of B. subtilis (GenBank TM accession number D10981), bovine (GenBank TM accession number M10543), and A. fulgidus (GenBank TM accession number AE000782) tRNA Trp (see Fig. 1 below). Individual pairs of the oligodeoxyribonucleotides were mixed and incubated at 60°C for 15 min to form double-stranded DNA fragments, which were separately inserted into the SfiI and HindIII restriction sites of the E. coli pGEM-9zf(Ϫ) vector (Promega) (4). PCRbased site-directed mutagenesis was employed to construct single and double mutations of N73 and the 1-72 base pairs using a proofreading Pfu DNA polymerase (Stratagene). All recombinant plasmids were obtained by 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-gal, Sigma Chemical Co.) Blue-White screening and confirmed by DNA sequencing using the BigDye sequencing kit (Applied Biosystems).

Constructs of Wild-type and Mutant tRNA
Sequence Alignment of the tRNA Trp Gene-Sequences of B. subtilis, A. fulgidus, and bovine tRNA Trp were aligned using the program Clust-alW. The position of each residue was assigned, and the cloverleaf structures of these three tRNA Trp were constructed (Fig. 1). The modified nucleotides in native B. subtilis (10) and bovine tRNA Trp (11) are known. The modified nucleotides of native A. fulgidus tRNA Trp have not been analyzed, but those of Halobacterium volcanii tRNA Trp were regarded as providing a first approximation description of the modifications in wild-type archaeal tRNA Trp (12,13).
Expression and Purification of tRNA Trp -Recombinant pGEM-9zf(Ϫ)-derived plasmids were transformed into the E. coli JM109 and cultured in M9-glycerol medium supplemented with 50 g/ml ampicillin, 1% glycerol, 0.01 mM FeCl 3 , 0.1 mM CaCl 2 , and 100 mg/liter thiamine, as described previously (4). To prepare the 15 N-labeled tRNA, 15 NH 4 Cl (Cambridge Isotope Laboratories, Inc.) was used as a nitrogen source in the M9-glycerol medium (14). When culture absorbance reached A 550 ϭ 0.16, 0.2 mM IPTG was added to induce the culture for 6 h. The cells were harvested by centrifugation, and total tRNA was isolated by phenol extraction (pH 6.4). To remove DNA and high molecular weigh RNA, the supernatant was incubated with 2.5 M NaCl at 4°C for 3 h. The total tRNA in the supernatant was purified on DEAE-Sepharose CL-6B (Sigma), and tRNA Trp was separated by reversephase HPLC on a Vydac C4-derivatized silica column (15). The fractions containing the tRNA Trp were identified by dot-blot hybridization, precipitated, and washed twice with 70% ethanol, stored at Ϫ20°C, and lyophilized before use.
Gel Electrophoresis of tRNA-12% denaturing polyacrylamide gel with 8 M urea was warmed up by pre-running to 50°C, and tRNA Trp preparations were incubated at 100°C for 10 min with loading buffer containing formamide (50% v/v) prior to electrophoresis in TBE electrophoresis buffer (90 mM Tris-borate, pH 8.0, 2 mM EDTA) at 1000 V for 6 h. The gel was stained with 0.1% toluidine blue (Sigma) in 7.5% acetic acid for 15 min, and destained with 2.5% acetic acid.
Identification of Modified Nucleotides-To analyze the modified nucleotides in cloned bovine and A. fulgidus tRNA Trp , each wild-type tRNA Trp (20 g) was hydrolyzed as described previously (16). Each nucleotide detected in the HPLC eluate was identified based on comparison of its UV spectrum and retention time with ribonucleoside reference standards. All modified nucleotides were determined in terms of moles of residue per mole of tRNA using Br 8 G as internal standard.
Cloning and Preparation of TrpRS-The TrpRS of B. subtilis hyperexpressed from recombinant plasmid pKSW1 was purified as described (17,18). A pBluescript SKϩ based cDNA clone with the human TrpRS gene (GenBank TM accession number NM_004184) inserted into an EcoRI site was obtained from J. Justesen (19); the full-length gene was digested by EcoRI and subcloned into pTrcHis-B expression vector (Invitrogen). The sequence between vector mini-cistron and initial ATG of human TrpRS gene, encoding His 6 -tag and a leading sequence, was removed by PCR-based site-directed mutagenesis using proofreading Pfu DNA polymerase prior to transformation into E. coli JM109 strain. The human TrpRS was induced by 1 mM IPTG and precipitated from the French press crude extract by addition of ammonium sulfate to 3 M. The pellet containing the synthetase was resuspended and applied to Phenyl-Sepharose 6 Fast Flow column followed by a Q-Sepharose Fast Flow column (both from Amersham Biosciences, Inc.). The fractions were screened for tryptophanylation activity using A. fulgidus wild-type tRNA Trp as substrate. The purified human TrpRS was concentrated to 0.5 mg/ml in 50% glycerol and stored at Ϫ20°C.
To obtain A. fulgidus TrpRS, a pUC18-derived cDNA clone (ATCC number, 629131; TIGR (The Institute for Genomic Research) locus, AF1694) containing TrpRS gene (trpS) (GenBank TM accession number AE000986) of A. fulgidus was supplied by ATCC. A pair of primers was designed to flank the A. fulgidus TrpRS gene with a BamHI site at the 5Ј-end and EcoRI site at the 3Ј-end, and proofreading Pfu DNA polymerase was used to yield the full-length A. fulgidus TrpRS gene. The PCR-amplified fragment was digested by BamHI and EcoRI and subcloned into the same sites of pTrcHis-A expression vector (Invitrogen). The recombinant expression vector was transformed into E. coli BL21-CodonPlus(DE3)-RIL cell (Stratagene). The His 6 -tagged A. fulgidus TrpRS was hyperexpressed by induction with 0.5 mM IPTG and extracted by French press. The crude extract containing soluble His 6tagged A. fulgidus TrpRS was mixed with 3 ml of nickel-nitrilotriacetic acid agarose slurry (Qiagen) by gently shaking at 4°C for 30 min. The mixture was packed in a mini column for gravity flow chromatography. After the resin was drained and washed with 10 mM imidazole until A 280 was constant and further washed with 20 mM imidazole. The enzyme was eluted with 100 mM imidazole. The fractions containing A. fulgidus TrpRS were concentrated and dialyzed by means of Centricon (Amicon) and stored at Ϫ20°C in 50% glycerol.  (10,11). On the A. fulgidus tRNA Trp structure, the tentative nucleotide modifications shown are those found in H. volcanii tRNA Trp (12,13). Nucleotides that are identical on the B. subtilis and A. fulgidus tRNA Trp sequences but dissimilar in the bovine tRNA Trp sequence are indicated as boxed letters in the B. subtilis tRNA Trp structure (a). Nucleotides that are identical in the bovine and A. fulgidus tRNA Trp sequences but dissimilar in the B. subtilis tRNA Trp sequences are indicated as boxed letters in bovine tRNA Trp structure (c). Nucleotides that are identical in the bovine and B. subtilis tRNA Trp sequence but dissimilar in the A. fulgidus tRNA Trp sequence are indicated as boxed letters in A. fulgidus tRNA Trp structure (b).

Tryptophanylation Kinetics-Tryptophanylation with B. subtilis
TrpRS was performed at 22°C as described (20). Tryptophanylation with human TrpRS was carried out at 30°C in reaction buffer containing 100 mM Tris-HCl pH 7.5, 10 mM magnesium acetate, with 2.4 mM ATP and 10 M L-[5-3 H]tryptophan (5 Ci/mmol, Amersham Biosciences, Inc.). Tryptophanylation with A. fulgidus TrpRS was carried out at 65°C in reaction buffer containing 100 mM MOPS (Sigma), pH 7.0, 2 mM dithiothreitol, 50 mM KCl, 20 mM MgCl 2 , and 100 mM NaCl with 2.4 mM ATP and 10 M L-[5-3 H]tryptophan (5 Ci/mmol, Amersham Biosciences, Inc.). At various time points, an aliquot of the reaction mixture was removed and spotted onto a 3MM (Whatman) filter. Filters were soaked in ice-cold 5% trichloroacetic acid followed by 95% ethanol wash before subjected to liquid scintillation counting and subtraction of control radioactivity from reaction mixture without added tRNA Trp . When the steady-state kinetic parameters k cat and K m for tRNA Trp were to be determined, a saturating concentration of 10 M L-[5-3 H]tryptophan (10 Ci/mmol, Amersham Pharmacia) for TrpRS was present in the assay mixture. The tRNA concentration was varied over a 10-fold range, all at least 100-fold in excess over TrpRS. Assays were performed in duplicates using six tRNA concentrations and two TrpRS concentrations, and errors on individual kinetic constants were consistently less than 15%. Kinetic parameters from three replicate experiments were estimated by fitting data into the Eadie-Hofstee plot using data analysis software Origin 6.1 (Originlab) and averaged.
NMR Spectroscopy-15 N-Labeled samples of bovine wild-type tRNA Trp and its A73G mutant were prepared as described previously (14). Sensitivity-enhanced gradient two-dimensional 1 H-and 15 N-HSQC spectra were performed on a Varian INOVA 500 NMR spectrometer at 30°C. Spectral widths at the proton and nitrogen dimensions were 12,000 and 6000 Hz, respectively.

RESULTS
Characterization of tRNA Trp from Three Domains-On the basis of tRNA Trp sequence alignment (Fig. 1 For all three recombinant tRNA Trp , base substitutions at N73 did not greatly influence the expression levels of the tRNA in E. coli. The bulk of bovine or A. fulgidus tRNA Trp was eluted in one major HPLC peak as revealed by dot-blot hybridization ( Fig. 2) in contrast to the multiple HPLC peaks observed with B. subtilis tRNA Trp differing in nucleotide modifications (15). Compared with B. subtilis tRNA Trp , bovine and A. fulgidus tRNA Trp was eluted earlier in the HPLC (Fig. 2), suggesting that the latter two tRNA molecules were less hydrophobic. Similar HPLC profiles were observed for all four N73 variants within each kind of tRNA Trp .
Compared with native bovine and H. volcanii tRNA Trp , the bovine and A. fulgidus tRNA Trp hyperexpressed in E. coli lacked some of the species-specific modifications, and acquired other modifications typical of host E. coli tRNA Trp (Table I).
The appearance of T in these two cloned tRNA molecules, possibly in place of 54 of native bovine tRNA Trp , and m 1 54 of archaeal tRNA Trp exemplified by H. volcanii tRNA Trp , was a striking difference between the cloned and native molecules, which likely arose from differences between E. coli and bovine or archaeal nucleoside-modifying enzymes. T is a highly conserved signature nucleotide in bacterial tRNA. Another difference was the presence in the cloned molecules of 4-thiouridine (s 4 U) (Table I), which is a signature modification at position 8 of E. coli tRNA but never observed in eukaryotic or archaeal tRNA (8). The appearance of 2Ј-O-methylcytidine (Cm) in cloned bovine tRNA Trp but not in cloned A. fulgidus tRNA Trp clearly indicates that the conversion of C to 2Ј-O-methylcytidine (Cm) depends not only on the availability of the requisite modifying enzyme system in E. coli host but also on appropriate tRNA sequence setting. It is also intriguing that no dihydrouridine (D) was observed in cloned bovine or A. fulgidus tRNA Trp . This residue is present in native bovine and B. subtilis tRNA Trp but absent from native H. volcanii tRNA Trp . Electrophoresis in denaturing 12% polyacrylamide gel containing 8 M urea yielded different migration rates for the three cloned tRNAs that were consistent with their different lengths, A. fulgidus tRNA Trp being the longest with 77 residues, B. subtilis tRNA Trp being the shortest with 74 residues, and bovine tRNA Trp being intermediate with 75 residues (Fig. 3).
Dot blot hybridization by means of tRNA-specific probes identified the bovine tRNA Trp peak at about 42 min in the HPLC elution profile and the slower emergence of A. fulgidus tRNA Trp at about 52 min. Because endogenous E. coli tRNA Trp was eluted at about 70 min, both of these cloned tRNA Trp isolated would contain little endogenous tRNA Trp .
Bacterial TrpRS Recognition-Previously it was found that the rate of tryptophanylation by B. subtilis TrpRS for the G73A mutant is some 10-fold decreased relative to the G73 wild-type (4), suggesting that the discriminator base is a major identity  Table II shows that the variation of k cat /K m with the nature of N73 was caused mostly by a change in k cat rather than K m : the K m for G73 wild-type and the G73A, G73C, and G73U mutants all fell within the range of 0.02-0.03 M, whereas their k cat varied over an 18-fold range.
In contrast, the discrimination by B. subtilis TrpRS against heterologous tRNA Trp entailed differentiation at both the k cat and K m levels. For example, comparing the k cat /K m values for the best tRNA substrate (B. subtilis G73) and worst tRNA substrate (bovine A73C), the former excelled over the latter by 357-fold, which was the combined result of an 8.5-fold (0.02/ 0.17) difference in K m and 42-fold difference (5.09/0.12) in k cat . Likewise, the k cat of A. fulgidus A73 tRNA Trp for this enzyme was one-ninth the k cat for B. subtilis wild-type G73, and its K m was also 6-fold that of the latter. Similarly the 79-fold greater catalytic efficiency toward B. subtilis G73 compared with bovine A73G arose from a 7.5-fold lower K m and a 10.6-fold higher k cat . Clearly, this enzyme is endowed with stringent selectivity not only toward N73 with a preference of GϾAϾUϾC but also toward the remainder portion of the tRNA molecule that varies with the preference of bacterial tRNAϾarchaeal tRNAϾeukaryotic tRNA.
Eukaryotic TrpRS Recognition-Purified recombinant human TrpRS (Fig. 4) charged most efficiently bovine, less efficiently A. fulgidus, and least efficiently B. subtilis tRNA Trp . However, the bias displayed by human TrpRS toward tRNA Trp from the three biological domains was somewhat less extreme than the B. subtilis enzyme, yielding a relative k cat /K m of 0.27 toward A. fulgidus tRNA Trp , and relative k cat /K m of 0.02 toward B. subtilis G73 wild-type tRNA Trp , improving to 0.082 when G73 in the latter molecule was mutated to A73. That the human TrpRS preferred archaeal to bacterial tRNA Trp was entirely in keeping with a closer phylogenetic relationship of eukarya to archaea than to bacteria.
The dependence of human enzyme on the discriminator base position was also less extreme compared with the bacterial enzyme. Mutation of A73 in bovine tRNA Trp to the worst case of G73 reduces its relative k cat /K m to 0.21. The preference of human TrpRS for AϾCϾUϾG at position 73 held for all three biological sources of tRNA, and was more moderate than the N73 preference of the B. subtilis enzyme. Studies using in vitro tRNA transcripts also indicated a preference of AϾG for the human enzyme (6).
Archaeal TrpRS Recognition-A. fulgidus is a sulfatereducing hyperthermophilic archaeon. Its His 6 -tagged TrpRS, upon hyperexpression and purification from E. coli, yielded a polypeptide of molecular mass ϳ50 kDa in SDSpolyacrylamide gel electrophoresis (Fig. 5). This enzyme acylated A. fulgidus, bovine, and B. subtilis tRNA Trp at 65°C but with k cat /K m values lower than those of human and B. subtilis TrpRS. The requirement of this enzyme for high temperature to be active was manifest in the fact that at 30°C it displayed no measurable activity even toward wild-type A. fulgidus tRNA Trp , in contrast to human TrpRS, which could readily acylate wild-type A. fulgidus tRNA Trp at 30°C. The low relatively k cat /K m values of the A. fulgidus enzyme could arise from its dependence on modified nucleotides present in native archaeal tRNA but missing from the cloned A. fulgidus tRNA Trp , which may contribute to tRNA thermostability at the assay temperature of 65°C. A. fulgidus TrpRS exhibited a same preference of AϾCϾUϾG as human TrpRS. Moreover, although it showed approximate 20% reactivity toward B. subtilis A73 tRNA Trp and less than 4% reactivity toward bovine A73 tRNA Trp , it retained for these heterologous tRNA substrates its preference pattern of AϾCϾUϾG. The comparable activities of A. fulgidus TrpRS toward bacterial tRNA Trp and eukaryotic tRNA Trp are in accord with an intermediate phylogenetic position of the archaea between bacteria and eukarya.
NMR Spectroscopy of Bovine A73G Mutant tRNA Trp -Cloned bovine tRNA Trp was efficiently tryptophanylated by human TrpRS with a k cat /K m of 1.12 ϫ 10 8 s Ϫ1 M Ϫ1 . When its discriminator base A73 was mutated, loss of catalytic efficiency occurred with the largest loss caused by mutation to G, stemming mostly from a 3-fold decrease in k cat and a 1.5-fold increase in K m (Table II).    (14). More recently a similar combination of mutagenesis and two-dimensional NMR has yielded assignments of most of the base pair resonances of cloned bovine wild-type tRNA Trp (Fig. 6a)   A73G mutant (Fig. 6b) in the 15 N HSQC spectra. 2 Comparison of these two 15 N HSQC spectra indicates that bovine A73G maintains almost intact the overall structure of A73 wild-type, without major changes in the resonances other than a marked change in the chemical shift for the G1-C72 base pair. Upon replacement of A73 by G73, the G1 resonance moved in the 1 H dimension from 11.75 to 12.15 ppm.
Synergism between A73 and G1-C72-In view of the conformational effect of A73G mutation on the G1-C72 base pair in bovine tRNA Trp , variants involving N73 and 1-72, the first base pair in the acceptor stem, were constructed, hyperexpressed, and analyzed for tryptophanylation kinetics. As shown in Fig. 7, G1-C72 and A73 are both important identity elements for human TrpRS. Mutating either A73 to G73, or G1-C72 to A1-U72, resulted in an approximate 4-fold reduction in activity. A double mutant carrying both G73 and A1-U72 suffered a synergistic 20-fold loss of tryptophanylation relative to the wild-type. Disruption of the G1-C72 base pair altogether by mutating G1 to A1 also brought about a 20-fold activity loss. These observations confirm the NMR finding of a structural linkage between N73 and 1-72. Together they suggest that N73 and G1-C72 constitute a structural domain that is crucial for productive interaction with TrpRS. This domain requires an intact 1-72 base pair and achieves maximum preference when it comprises A73 and G1-C72.

DISCUSSION
Functional Effect of N73 Substitution-For B. subtilis tRNA Trp , the discriminator base G73 and the anticodon represent two major identity elements responsible for effective recognition by B. subtilis TrpRS, with A1-U72, G5-C68, and A9 also serving as minor identity elements. Of these elements, the role of the discriminator base is particularly intriguing: When G73 is mutated to A73, the tRNA Trp loses much of its reactivity with bacterial TrpRS but becomes active with eukaryotic yeast TrpRS. Such reduction of tryptophanylation caused by a mutation of discriminator base has been confirmed for B. subtilis and human TrpRS acting on in vitro tRNA Trp transcripts (6). In known tRNA Trp sequences from different species of the three biological domains (8), the discriminator base is overwhelmingly occupied by a purine: G73 in 19 bacteria, 13 chloroplasts, 95 mitochondria, and one eukaryotic cytoplasm (Toxoplasma gondoii); A73 in 13 eukaryotic cytoplasms, 6 archaea, and 13 mitochondria. The exceptions to this A/G dominance are U73 in Arbacia lixula mitochondria and the eukaryotic Dictyostelium discoideum cytoplasm, and C73 in Herpesvirus. The fact that bacterial TrpRS prefers G73, whereas eukaryotic TrpRS prefers A73, is thus entirely in accord with this remarkable G73/ A73 sequence dichotomy between bacteria and chloroplasts, on the one hand, and eukarya and archaea on the other. In the present study, the evolutionary sequence dichotomy is found to be mirrored by a corresponding enzyme-specificity dichotomy.
B. subtilis TrpRS displayed a strong a preference for G73 on B. subtilis wild-type tRNA Trp followed by A (relative k cat /K m of 0.11), and C and U trailing with about half the activity of A. This k cat /K m the preference is exercised mostly at the k cat rather than K m level. Because proofreading is not important for this enzyme (20), the dependence of k cat on G at position 73 is in accord with the finding using stopped-flow fluorometry and that the N73 of E. coli tRNA Trp contributes to the stability of the Trp-tRNA transition state during the transfer of activated Trp to the tRNA (21).
The archaeal enzyme A. fulgidus TrpRS acting on cloned A. fulgidus tRNA Trp preferred A73, followed by C73, U73, and G73. This is consistent with the exclusive occupation of the discriminator base by A in archaeal tRNA Trp . Because C73 ranks second as an effective discriminator nucleotide for archaeal TrpRS, it is therefore not important for this enzyme that the discriminator base position is a purine. Instead, an amino group on the six-member heterocyclic ring in discriminator base, which is common to A and C (at position 6 in A, or 2 in C), but unshared by U or G, might be more important for efficient recognition by this synthetase.
Human TrpRS acting on hyperexpressed bovine tRNA Trp was severalfold more active with A73 than with, in order of descending preference, C73, U73, and G73. The distinct preference of human TrpRS for A73 stands in total agreement with the overwhelming occurrence of A73 in eukaryotic tRNA Trp . The preference of C over U and G again suggests the possible importance of an amino group on the discriminator base.
Independent Action of Identity Elements in tRNA Trp -Aminoacyl-tRNA synthetases are often guided by multiple identity elements on its cognate tRNA substrate. This is certainly the case with TrpRS from all three biological domains. Each of these TrpRS displays (i) defined preference toward N73 and (ii) defined preference toward the rest of the tRNA Trp molecule structure in which N73 is located. For the three TrpRS examined, their preferences based on k cat /K m values (Table II)  The Type I and Type II preferences act independently. For example, because of the Type II preference, B. subtilis TrpRS is catalytically more efficient acting on all four N73 forms of homologous tRNA Trp from B. subtilis than any form of heterologous bovine or archaeal tRNA Trp regardless of the nature of the discriminator base. Nonetheless, its Type I preference of GϾAϾUϾC remains evident among the N73 forms within each of these three biological sources of tRNA Trp . A. fulgidus TrpRS is more active toward even the A73G of A. fulgidus tRNA Trp than any of the four forms of bovine tRNA Trp ; human TrpRS is also more active toward even the A73G of bovine tRNA Trp than any of the four forms of B. subtilis tRNA Trp . Yet these two enzymes retain their AϾCϾUϾG preference among tRNA Trp from every domain.
The Type II preferences are fully consistent with an intermediate position occupied by archaea between the phylogenetic divergence of bacteria and eukarya. Fig. 1 shows that in A. fulgidus tRNA Trp , 18 residues (23% of all residues) are identical to corresponding residues in bovine tRNA Trp but unshared by B. subtilis tRNA Trp . At the same time, 18 other residues in A. fulgidus tRNA Trp are identical to corresponding residues in B. subtilis tRNA Trp but unshared by bovine tRNA Trp . In contrast, merely four residues are common to the bacterial and eukaryotic tRNA Trp but unshared by archaeal tRNA Trp . These sequence identities therefore strongly confirm the observed Type II kinetic preferences. They point to a wide divergence between bacteria and eukarya, with archaea occupying a phylogenetic position intermediate between them and displaying unmistakable tRNA Trp sequence similarity to both of them.
Influence of A73 on G1-C72 in Bovine tRNA Trp -To examine the effects of N73 on tRNA structure that might be significant to its role as an identity element, a structural comparison of the bovine wild-type A73 tRNA Trp with its A73G mutant was performed by solution NMR spectroscopy. The mutation was found to cause few NMR spectral changes except for a chemical shift in the resonance of the G1-C72 base pair. This indicates the occurrence of a conformational change in the microenvironment of this base pair caused by the A73G mutation. This finding is consistent with the suggestions that N73 of tRNA can base-stack on the nearby first base pair of the tRNA acceptor stem helix and extend the stacking in this stem (22)(23)(24) and that N73 plays an important role in the stabilization of the acceptor stem helix by the single-stranded 3Ј terminus (25). Thus the conformational change in G1-C72 in bovine tRNA Trp elicited by the A73G mutation, as revealed by NMR, could be due to an effect on the base stacking between G73 and G1-C72. For reaction with human TrpRS, the A1-U72 and A73G mutations each induced a comparable 4-fold loss of activity. When the A73G and A1-U72 mutations were introduced together into bovine tRNA Trp , a ϳ20-fold loss of reaction rate resulted. Disruption of the 1-72 base pair by mutating G1 to A1 likewise caused a ϳ25-fold loss (Fig. 7). Therefore the A73 and G1-C72 elements clearly interacted to form a key sub-region on the tRNA. The maintenance of an optimal conformation in this sub-region near the acceptor terminus of the tRNA could be important for optimal interaction with eukaryotic TrpRS.
In conclusion, the TrpRS-tRNA Trp recognition process is extraordinary in that an identity-element divergence, in the form of a G73/A73 dichotomy, divides the bacterial and archaealeukaryotic phylogenetic domains. Although the origin and evolutionary significance of this surprising dichotomy are far from understood, this dichotomy itself provides unique advantages for enquiry into the nature of genetic code and tRNA evolution. Evidence for an intermediate phylogenetic position of archaea between bacteria and eukarya, as revealed by both TrpRS kinetics and tRNA Trp sequences, the independent action of different identity elements on tRNA, and the functional linkage in bovine tRNA Trp between the discriminator base and the first base pair of the acceptor stem for recognition by TrpRS, represents only initial examples of the rich harvest of insight that may be expected from this remarkable system.