3种水稻线粒体tRNA~(Trp)突变体的克隆和活力鉴定

 设计并完成了 3种水稻线粒体tRNATrp的突变 ,体外转录并用枯草杆菌和人色氨酰tRNA合成酶 (TrpRS)对tRNATrp及其突变体进行了活力测定 .3种突变体的氨酰化活力比野生型水稻线粒体tRNATrp分别上升了 1 8、1 5和 5倍 .说明A1 U72和G5 C68对于提高线粒体tRNATrp被细胞质TrpRS氨酰化能力的作用并不大 ,细胞质tRNATrp与细胞质TrpRS的识别方式并不适用于线粒体tRNATrp与细胞质TrpRS的相互识别 .研究结果对于了解线粒体tRNATrp和细胞质TrpRS的相互识别及药物设计有重要意义     

Residues Lys-149 and Glu-153 switch the aminoacylation of tRNA(Trp) in Bacillus subtilis

Tryptophanyl-tRNA synthetase (TrpRS) consists of two identical subunits that induce the cross-subunit binding mode of tRNA(Trp). It has been shown that eubacterial and eukaryotic TrpRSs cannot efficiently cross-aminoacylate the corresponding tRNA(Trp). Although the identity elements in tRNA(Trp) that confer the species-specific recognition have been identified, the corresponding elements in TrpRS have not yet been reported. In this study two residues, Lys-149 and Glu-153, were identified as being crucial for the accurate recognition of tRNA(Trp). These residues reside adjacent to the binding pocket for Trp-AMP and show phylogenic diversities in the charge on their side chains between eubacteria and eukaryotes. Single mutagenesis at Lys-149 or Glu-153 reduced the activity of TrpRS in the activation of Trp. The reduction was less than that caused by the double mutant WBHA (K149D/E153R). It is unusual that E153G had no detectable activity in the activation of Trp unless tRNA(Trp) was added to the reaction. In addition, we successfully switched the species specificity of Bacillus subtilis TrpRS recognition of tRNA(Trp). The affinity of WBHA, K149E and E153K to human tRNA(Trp) was 31-, 13.5-, and 12.9-fold greater than that of wild type B. subtilis TrpRS, respectively. Indeed WBHA and E153K were found to prefer genuine human tRNA(Trp) to their cognate eubacteria tRNA(Trp).     

Structures of tryptophanyl-tRNA synthetase II from Deinococcus radiodurans bound to ATP and tryptophan. Insight into subunit cooperativity and domain motions linked to catalysis

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.     

枯草杆菌TrpRS的固定化及亲和RNA筛选

为研究 t RNATrp与色氨酰 - t RNA合成酶 ( Trp RS)的相互识别及其结构与功能的关系 ,纯化了枯草杆菌 Trp RS,并用溴化氰活化的 Sepharose4B将 Trp RS固定化 ,固定化 Trp RS的蛋白回收率为 95.5% ,活力回收率为 31 .3% .研究了固定化 Trp RS的酶学性质 ,其热稳定性和贮存稳定性方面均比液相 Trp RS有了较大的提高 ,最适温度、最适 p H均有一定程度的增大 ,工作稳定性良好 .以固定化 Trp RS为亲和层析介质 ,对含有 2 0个核苷酸随机序列 ,长度为 56个核苷酸的单链RNA随机库进行了三轮筛选 .实验结果表明 ,固定化 Trp RS可以作为 SELEX亲和层析介质 ,进行模拟 t RNATrp分子的 RNA随机库的 SELEX筛选 .     

Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp 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. Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.     

固定化枯草杆菌色氨酰tRNA合成酶(英文)

为研究ｔＲＮＡＴｒｐ 与色氨酰ｔＲＮＡ合成酶（ＴｒｐＲＳ） 的相互识别及其结构、功能关系, 纯化了枯草杆菌ＴｒｐＲＳ并用溴化氰活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ 将ＴｒｐＲＳ固定化, 固定化ＴｒｐＲＳ的蛋白质回收率为９５ ．５ ％ , 活力回收率为３１．３％ 。研究了固定化ＴｒｐＲＳ的酶学性质, 其热稳定性和贮存稳定性方面均比液相ＴｒｐＲＳ有了较大的提高, 最适温度、最适ｐＨ 均有一定程度的增大, 工作稳定性良好。以固定化ＴｒｐＲＳ为亲和层析介质, 对含有２０ 个核苷酸随机序列、长度为５６ 个核苷酸的单链ＲＮＡ 随机库进行了３ 轮筛选,ＲＮＡ 群体亲和固定化ＴｒｐＲＳ的比例从４ ．３ ％ 上升至１４ ．７ ％ 。筛选得到了与ｔＲＮＡＴｒｐ 氨基酸接受茎类似的ＲＮＡ二级结构。实验结果表明固定化ＴｒｐＲＳ可以作为ＳＥＬＥＸ 亲和层析介质, 进行模拟ｔＲＮＡＴｒｐ 分子的ＲＮＡ 随机库的ＳＥＬＥＸ 筛选。     

Evidence for annexin II-S100A10 complex and plasmin in mobilization of cytokine activity of human TrpRS

In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.     

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 A and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzyme's apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.     

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.     

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.     

Oxidative stress-responsive intracellular regulation specific for the angiostatic form of human tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase (TrpRS) exists in two forms in human cells, i.e., a major form which represents the full-length protein and a truncated form (mini TrpRS) in which an NH(2)-terminal extension is deleted because of alternative splicing of its pre-mRNA. Mini TrpRS can act as an angiostatic factor, while full-length TrpRS is inactive. We herein show that an oxidized form of human glyceraldehyde-3-phosphate dehydrogenase (GapDH) interacts with both full-length and mini TrpRSs and specifically stimulates the aminoacylation potential of mini, but not full-length, TrpRS. In contrast, reduced GapDH did not bind to TrpRSs and did not influence their aminoacylation activity. Mutagenesis experiments clarified that the NH(2)-terminal Rossmann fold region of GapDH is crucial for its interaction with mini TrpRS as well as tRNA and for the regulation of its aminoacylation potential and suggested that monomeric GapDH can bind to mini TrpRS and stimulate its aminoacylation activity. These results suggest that the angiostatic human mini, but not the full-length, TrpRS may play an important role in the intracellular regulation of protein synthesis under conditions of oxidative stress.     

2.9 A crystal structure of ligand-free tryptophanyl-tRNA synthetase: domain movements fragment the adenine nucleotide binding site

The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 A using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5'AMP complex (TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal alphaA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189-1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997     

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.     

A Concerted Tryptophanyl-adenylate-dependent Conformational Change inBacillus subtilisTryptophanyl-tRNA Synthetase Revealed by the Fluorescence of Trp92

A semi-conserved tryptophan residue ofBacillus subtilistryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNA^Trpbinding and recognition. The crystal structure of theBacillus stearothermophilusTrpRS tryptophanyl-5′-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy.B. subtilisTrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5′-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNA^Trpis unlikely.Time-resolved fluorescence indicates conformational heterogeneity ofB. subtilisTrp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed- TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure.The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an α-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 Å) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local α-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small α-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.     

14 What RNA world ?? Ancestral polypeptides likely participated in the origins of translation

A widespread consensus holds that protein synthesis according to a genetic code was launched entirely by sophisticated RNA molecules that played both coding and functional roles. This belief persists, unsupported by phylogenetic evidence for ancestral ribozymes that catalyzed either amino acid activation or tRNA aminoacylation. By contrast, we have adduced strong experimental evidence that the most highly conserved portions of contemporary aminoacyl-tRNA synthetases (aaRS) accelerate both reactions well in excess of rates achieved by RNA aptomers derived from combinatorial libraries and of rates required for primordial protein synthesis. Such ancestral enzymes, or “Urzymes”, characterized for Class I (TrpRS (Pham et al., 2010, 2007) and LeuRS (Collier et al., 2013); 130 residues) and Class II (HisRS; 120–140 residues; (Li et al., 2011)) synthetases generally have promiscuous amino acid specificities, whereas ATP and cognate tRNA affinities are within an order of magnitude of those for contemporary enzymes. These characteristics match or exceed expectations for the primordial catalysts necessary to launch protein synthesis. Structural hierarchies in Class I and II aaRS also exhibit plateaus of increasing enzymatic activity, suggesting that catalysis by peptides similar to the Aleph motif identified by Trifonov (Sobolevsky et al.) may have been both necessary and sufficient to launch protein synthesis. Sense/antisense alignments of TrpRS and HisRS Urzyme coding sequences reveal unexpectedly high middle-base complementarity that increases in reconstructed ancestral nodes (Chandrasekaran et al.), consistent with the proposal of Rodin and Ohno (Rodin & Ohno, 1995). Thus, these ancestors were likely coded by opposite strands of the same gene, favoring simultaneous expression of aaRS activating both hydrophobic (core) and hydrophilic (surface) amino acids. Our results support the view that aaRS coevolved with cognate tRNAs from a much earlier stage than that envisioned under the RNA World hypothesis, and that their descendants make up appreciable portions of the proteome.     

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.