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Throughout three domains of life, alanyl-tRNA synthetases (AlaRSs) recognize a G3:U70 base pair in the acceptor stem of tRNA Ala as the major identity determinant of tRNA Ala The crystal structure of the archaeon Archaeoglobus fulgidus AlaRS in complex with tRNA Ala provided the basis for G3:U70 recognition with residues (Asp and Asn) that are conserved in the three domains [Naganuma M, et al. (2014) Nature 510:507-511]. The recognition mode is unprecedented, with specific accommodation of the dyad asymmetry of the G:U wobble pair and exclusion of the dyad symmetry of a Watson-Crick pair. With this conserved mode, specificity is based more on "fit" than on direct recognition of specific atomic groups. Here, we show that, in contrast to the archaeal complex, the Escherichia coli enzyme uses direct positive (energetically favorable) minor groove recognition of the unpaired 2-amino of G3 by Asp and repulsion of a competing base pair by Asn. Strikingly, mutations that disrupted positive recognition by the E. coli enzyme had little or no effect on G:U recognition by the human enzyme. Alternatively, Homo sapiens AlaRS selects G:U without positive recognition and uses Asp instead to repel a competitor. Thus, the widely conserved Asp-plus-Asn architecture of AlaRSs can select G:U in a straightforward (bacteria) or two different unconventional (eukarya/archaea) ways. The adoption of different modes for recognition of a widely conserved G:U pair in alanine tRNAs suggests an early and insistent role for G:U in the development of the genetic code., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg 2+ , were approximately 50%, 60% and 80%, respectively. Mg 2+ also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg 2+ , were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg 2+ ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The N¹-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m¹G37 positively regulates the aminoacylation of the tRNA, and simultaneously functions to prevent the +1 frameshift on the ribosome. Interestingly, Trm5 and TrmD have completely distinct origins, and therefore bear different tertiary folds. In this review, we describe the different strategies utilized by Trm5 and TrmD to recognize their substrate tRNAs, mainly based on their crystal structures complexed with substrate tRNAs.

Proteins with knotted configurations, in comparison with unknotted proteins, are restricted in conformational space. Little is known regarding whether knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. TrmD is a bacterial methyltransferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product, m 1 G37-tRNA, is essential for life and maintains protein-synthesis reading frames. Using an integrated approach of structural, kinetic, and computational analysis, we show that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot undergoes complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding, thereby stabilizing tRNA binding and allowing assembly of the active site. This work demonstrates new principles of knots as organized structures that capture the free energies of substrate binding and facilitate catalysis.

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal.

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

Post-transcriptional modifications of bases within the transfer RNAs (tRNA) anticodon significantly affect the decoding system. In bacteria and eukaryotes, uridines at the wobble position (U34) of some tRNAs are modified to 5-methyluridine derivatives (xm⁵U). These xm⁵U34-containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box. In Escherichia coli (E.coli), the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine (cmnm⁵U) to 5-methylaminomethyl uridine (mnm⁵U). The C-terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)-dependent deacetylation of cmnm⁵U to 5-aminomethyl uridine (nm⁵U), whereas the N-terminal domain (MnmC2) catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of nm⁵U, leading to the final product, mnm⁵U34. Here, we determined the crystal structure of E.coli MnmC containing FAD, at 3.0 Å resolution. The structure of the MnmC1 domain can be classified in the FAD-dependent glutathione reductase 2 structural family, including the glycine oxidase ThiO, whereas the MnmC2 domain adopts the canonical class I methyltransferase fold. A structural comparison with ThiO revealed the residues that may be involved in cmnm⁵U recognition, supporting previous mutational analyses. The catalytic sites of the two reactions are both surrounded by conserved basic residues for possible anticodon binding, and are located far away from each other, on opposite sides of the protein. These results suggest that, although the MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner., (Copyright © 2011 The Protein Society.)

tRNA precursors undergo a maturation process, involving nucleotide modifications and folding into the L-shaped tertiary structure. The N1-methylguanosine at position 37 (m1G37), 3' adjacent to the anticodon, is essential for translational fidelity and efficiency. In archaea and eukaryotes, Trm5 introduces the m1G37 modification into all tRNAs bearing G37. Here we report the crystal structures of archaeal Trm5 (aTrm5) in complex with tRNA(Leu) or tRNA(Cys). The D2-D3 domains of aTrm5 discover and modify G37, independently of the tRNA sequences. D1 is connected to D2-D3 through a flexible linker and is designed to recognize the shape of the tRNA outer corner, as a hallmark of the completed L shape formation. This interaction by D1 lowers the K(m) value for tRNA, enabling the D2-D3 catalysis. Thus, we propose that aTrm5 provides the tertiary structure checkpoint in tRNA maturation.

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2'-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 A resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal beta-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the lambda form as its substrate.

tmRNA and small protein B (SmpB) are essential trans-translation system components. In the present study, we determined the crystal structure of SmpB in complex with the entire tRNA domain of the tmRNA from Thermus thermophilus. Overall, the ribonucleoprotein complex (tRNP) mimics a long-variable-arm tRNA (class II tRNA) in the canonical L-shaped tertiary structure. The tmRNA terminus corresponds to the acceptor and T arms, or the upper part, of tRNA. On the other hand, the SmpB protein simulates the lower part, the anticodon and D stems, of tRNA. Intriguingly, several amino acid residues collaborate with tmRNA bases to reproduce the canonical tRNA core layers. The linker helix of tmRNA had been considered to correspond to the anticodon stem, but the complex structure unambiguously shows that it corresponds to the tRNA variable arm. The tmRNA linker helix, as well as the long variable arm of class II tRNA, may occupy the gap between the large and small ribosomal subunits. This suggested how the tRNA domain is connected to the mRNA domain entering the mRNA channel. A loop of SmpB in the tRNP is likely to participate in the interaction with alanyl-tRNA synthetase, which may be the mechanism for the promotion of tmRNA alanylation by the SmpB protein. Therefore, the tRNP may simulate a tRNA, both structurally and functionally, with respect to aminoacylation and ribosome entry.

Glutamyl-tRNA synthetase (GluRS) is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation. We analyzed the role of tRNA in amino acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the amino acid-binding site, which is defective and accounts for the binding of incorrect amino acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the amino acid activation.

An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.

Kasugamycin (Ksg) specifically inhibits translation initiation of canonical but not of leaderless messenger RNAs. Ksg inhibition is thought to occur by direct competition with initiator transfer RNA. The 3.35-A structure of Ksg bound to the 30S ribosomal subunit presented here provides a structural description of two Ksg-binding sites as well as a basis for understanding Ksg resistance. Notably, neither binding position overlaps with P-site tRNA; instead, Ksg mimics codon nucleotides at the P and E sites by binding within the path of the mRNA. Coupled with biochemical experiments, our results suggest that Ksg indirectly inhibits P-site tRNA binding through perturbation of the mRNA-tRNA codon-anticodon interaction during 30S canonical initiation. In contrast, for 70S-type initiation on leaderless mRNA, the overlap between mRNA and Ksg is reduced and the binding of tRNA is further stabilized by the presence of the 50S subunit, minimizing Ksg efficacy.

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3' termini. Its crystal structure has revealed a four-layer alphabeta/betaalpha sandwich fold that is typically found in the metallo-beta-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.

2-Thioribothymidine (s(2)T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s(2)T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s(2)T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s(2)T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s(2)T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro.

In the final step of tRNA splicing, the 2'-phosphotransferase catalyzes the transfer of the extra 2'-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2'-phosphotransferase protein from Aeropyrum pernix K1 at 2.8 Angstroms resolution. The structure of the 2'-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2'-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2'-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2'-phosphotransferase.

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Nucleoside derivatives of 2-amino-6-(2-thienyl)purine (s) are fluorescent and can be site-specifically incorporated into RNA by transcription mediated by unnatural base pairs between s and its complementary bases. To utilize the fluorescent s base as a probe, we examined the fluorescent properties of s in DNA and RNA fragments. The nucleoside of s exhibited a fluorescence emission centered at 432 nm, characterized by two major excitation maxima (299 and 352 nm), and its quantum yield was 0.41 at pH 7.0. The fluorescence intensity of s in DNA fragments (12-mer) differed depending on circumstances such as the base components, stacking, and pairings. The s base was introduced into a tRNA at specific positions by specific transcription using the unnatural base pairs. The fluorescence intensity of s at each position reflected the local structural features of the tRNA molecules.

During the first step of porphyrin biosynthesis in Archaea, most bacteria, and in chloroplasts glutamyl-tRNA reductase (GluTR) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Elements in tRNA(Glu) important for utilization by Escherichia coli GluTR were determined by kinetic analysis of 51 variant transcripts of E. coli Glu-tRNA(Glu). Base U8, the U13*G22**A46 base triple, the tertiary Watson-Crick base pair 19*56, and the lack of residue 47 are required for GluTR recognition. All of these bases contribute to the formation of the unique tertiary core of E. coli tRNA-(Glu). Two tRNA(Glu) molecules lacking the entire anticodon stem/loop but retaining the tertiary core structure remained substrates for GluTR, while further decreasing tRNA size toward a minihelix abolished GluTR activity. RNA footprinting experiments revealed the physical interaction of GluTR with the tertiary core of Glu-tRNA(Glu). E. coli GluTR showed clear selectivity against mischarged Glu-tRNA(Gln). We concluded that the unique tertiary core structure of E. coli tRNA(Glu) was sufficient for E. coli GluTR to distinguish specifically its glutamyl-tRNA substrate.

Many tRNA molecules that recognize the purine-ending codons but not the pyrimidine-ending codons have a modified uridine at the wobble position, in which a methylene carbon is attached directly to position 5 of the uracil ring. Although several models have been proposed concerning the mechanism by which the 5-substituents regulate codon-reading properties of the tRNAs, none could explain recent results of the experiments utilizing well-characterized modification-deficient strains of Escherichia coli. Here, we first summarize previous studies on the codon-reading properties of tRNA molecules with a U derivative at the wobble position. Then, we propose a hypothetical mechanism of the reading of the G-ending codons by such tRNA molecules that could explain the experimental results. The hypothesis supposes unconventional base pairs between a protonated form of the modified uridines and the G at the third position of the codon stabilized by two direct hydrogen bonds between the bases. The hypothesis also addresses differences between the prokaryotic and eukaryotic decoding systems.

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.

The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.

Proteins with unnatural amino acids at specific positions can be produced through cell-free protein synthesis. The synthesis of such molecules can, in principle, be facilitated by improving the codon reading efficiency of the tRNA that inserts the unnatural amino acid. In the present study, we prepared tRNA molecules with 2'-O-methyl nucleosides at the second and third positions of the anticodon and measured their codon-reading efficiencies. The results indicated, contrary to our expectation, that the modification damaged the decoding function completely.

The cell-free system for biosynthesis of proteins is becoming an important tool for protein engineering. In particular, introduction of the unnatural amino acids is achieved though cell-free protein synthesis with the use of chemically acylated tRNA that recognizes a specific codon. In the original method, however, it was difficult to control the system through changing tRNA composition, as the endogenous tRNAs are involved in the reaction. Thus, in the present study, we digested the tRNA within Escherichia coli S30 extract with resin-bound RNase A, and estimated the protein synthesis activity. It was revealed that this digestion process does not damage the activity, if a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), is present in the digestion reaction.

Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis. We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon. One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U). These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated. The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon.

RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial synthetase, which was needed for expression of the operational RNA code. The second synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of synthetases was added with the anticodon-containing domain of tRNAs.

In order to elucidate roles of the 2'-O-methylation of pyrimidine nucleotide residues of tRNAs, conformations of 2'-O-methyluridylyl(3'----5')uridine (UmpU), 2'-O-methyluridine 3'-monophosphate (Ump), and 2'-O-methyluridine (Um) in 2H2O solution were analyzed by one- and two-dimensional proton NMR spectroscopy and compared with those of related nucleotides and nucleoside. As for UpU and UmpU, the 2'-O-methylation was found to stabilize the C3'-endo form of the 3'-nucleotidyl unit (Up-/Ump-moiety). This stabilization of the C3'-endo form is primarily due to an intraresidue effect, since the conformation of the 5'-nucleotidyl unit (-pU moiety) was only slightly affected by the 2'-O-methylation of the 3'-nucleotide unit. In fact even for Up and Ump, the 2'-O-methylation significantly stabilizes the C3'-endo form by 0.8 kcal/.mol-1. By contrast, for nucleosides (U and Um), the C3'-endo form is slightly stabilized by 0.1 kcal/.mol-1. Accordingly, the stabilization of the C3'-endo form by the 2'-O-methylation is primarily due to the steric repulsion among the 2-carbonyl group, the 2'-O-methyl group and the 3'-phosphate group in the C2'-endo form. For some tRNA species, 2-thiolation of pyrimidine residues is found in positions where the 2'-O-methylation is found for other tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso-N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D-stem and/or in the anticodon stem.

Conformational characteristics of N4-acetyl-2'-O-methylcytidine (ac4Cm), 5-methyl-2'-O-methylcytidine (m5Cm) and N2-dimethyl-2'-O-methylguanosine (m2(2)Gm) found in tRNAs from extremely thermophilic archaebacteria were analyzed by proton NMR spectroscopy. The 2'-O-methylation of ac4C, m5C and m2(2)G was found to stabilize the C3'-endo form and therefore cause "conformational rigidity". In particular, the ac4Cm was found to be extremely rigid due to additive effects of the N4-acetylation and 2'-O-methylation. Therefore, tRNAs from the extremely thermophilic archaebacteria use the base modifications in combination with the 2'-O-methylation, resulting in stabilization of the A-type conformation at specific positions in the tRNAs even at very high temperatures. In contrast, mesophile tRNAs use for a given site only one of these ribose and base modifications each of which is effective enough by itself at ordinary temperatures. These findings are consistent with our previous findings that roles of a variety of post-transcriptional modifications are to regulate the conformational rigidity/flexibility which is essential for the tRNA functions.

The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.

By analyzing aminoacylation activities of variants of isoleucine tRNAs and glutamic acid tRNA from Escherichia coli, it was found that the anticodons are the major determinants for "identities" of these tRNAs. It was also shown that the post-transcriptional modifications are essential to aminoacylation of these tRNAs. Interactions between the tRNAs and the aminoacyl-tRNA synthetases were analyzed by NMR spectroscopy.

By two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, we analyzed dynamic structures of various tRNA species from Escherichia coli and Bacillus subtilis. Proton resonances due to the anticodon of the tRNA molecules were unambiguously identified by NOESY and 2D-HOHAHA techniques. Thus, it was found that rigidity/flexibility of the two types of modified uridines in the first position of the anticodon were certainly related with the codon recognition properties of the tRNA species.

1H-NMR analyses have been made on the conformations of 2-thioribothymidine (s2T), 2-thiodeoxyribothymidine (s2dT), as well as ribothymidine (T) and deoxyribothymidine (dT). s2T and s2dT exclusively take the anti form rather than the syn form. The C3'-endo-gg form of the sugar moiety is remarkably stabilized on modification of T to s2T, but not on modification of dT to s2dT. The steric effects of the 2-thiocarbonyl group and the 2'-hydroxyl group cause the rigidity of the C3'-endo-gg form of s2T. Such rigidity of s2T probably contributes to the thermostability of 2-thiopyrimidine polyribonucleotides and extreme thermophile tRNAs.

Proton NMR analyses have been made to elucidate the conformational characteristics of modified nucleotides as found in the first position of the anticodon of tRNA [derivatives of 5-methyl-2-thiouridine 5'-monophosphate (pxm5s2U) and derivatives of 5-hydroxyuridine 5'-monophosphate (pxo5U)]. In pxm5s2U, the C3'-endo form is extraordinarily more stable than the C2'-endo form for the ribose ring, because of the combined effects of the 2-thiocarbonyl group and the 5-substituent. By contrast, in pxo5U, the C2'-endo form is much more stable than the C3'-endo form, because of the interaction between the 5-substituent and the 5'-phosphate group. The enthalpy differences between the C2'-endo form and the C3'-endo form have been obtained as 1.1, -0.7, and 0.1 kcal/mol (1 cal = 4.184 J) for pxm5s2U, pxo5U, and unmodified uridine 5'-monophosphate, respectively. These findings lead to the conclusion that xm5s2U in the first position of the anticodon exclusively takes the C3'-endo form to recognize adenosine (but not uridine) as the third letter of the codon, whereas xo5U takes the C2'-endo form as well as the C3'-endo form to recognize adenosine, guanosine, and uridine as the third letter of the codon on ribosome. Accordingly, the biological significance of such modifications of uridine to xm5s2U/xo5U is in the regulation of the conformational rigidity/flexibility in the first position of the anticodon so as to guarantee the correct and efficient translation of codons in protein biosynthesis.

tRNA species from an extreme thermophile T. thermophilus that grows up to 85 degrees C have been found to be more thermostable than those from moderate thermophiles and mesophiles. Such thermostability of T. thermophilus tRNA species is partly due to the high contents of G.C base pairs in the stem regions. In addition, a novel modified nucleoside s2T has been found that substitutes T in position 54. The extent of 2-thiolation of T(54) has been found to depend on environmental temperatures from 50 to 80 degrees C. Two tRNA(Ile) species have been isolated from T. thermophilus HB8, tRNA(1aIle) with s2T(54) and tRNA(1bIle) with T(54), which have the identical nucleotide sequence except for position 54. However, the melting temperature of tRNA(1aIle) is higher by 3 degrees C than that of tRNA(1bIle). This clearly indicates that the 2-thiolation of T(54) contributes directly to the thermostability of T. thermophilus tRNA species. Proton NMR analyses have shown that the nucleoside s2T is "rigid" and predominantly takes the C3'-endo-gg-anti form of A-RNA, because of the steric effect of the bulky 2-thiocarbonyl groups and the 2'-hydroxyl group. Thus, the inherent rigidity of s2T in position 54 significantly enhances the stability of the tertiary structure of tRNA. In protein synthesis of T. thermophilus, s2T(54)-bearing tRNA and T(54)-bearing tRNA species are selectively utilized depending on environmental temperature. In the anticodons of major tRNA species from T. thermophilus, G or C exclusively appears in the first position, and GGN and CCN are favored over synonymous GCN or CGN. These characteristic anticodon sequences correspond to the characteristic codon usage in thermophile genes.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

The conformational characteristics of a modified nucleoside 4-acetylcytidine (ac4C) was analyzed by proton nuclear magnetic resonance spectroscopy. This nucleoside was found to take the C3'-endo form predominantly. The enthalpy difference between the C2'-endo form and the C3'-endo form of the ribose moiety of ac4C was determined and 4-acetylation was found to stabilize the C3'-endo form further by 0.83 kcal.mol-1. This conformational characteristics of ac4C is probably important for the correct codon recognition and the stability of the tertiary structure of tRNA molecule.

The interactions of tRNA species with aminoacyl-tRNA synthetases and polypeptide chain elongation factor Tu from Thermus thermophilus HB8 were studied by the analyses mainly of the circular dichroism band of 2-thioribothymidine in position 54 of T. thermophilus tRNA species.

A novel modified nucleoside located in the first position of the anticodon of yeast tRNAVal2a was isolated and its chemical structure was characterized as 5-carbamoylmethyluridine by means of ultraviolet absorption spectrum, mass spectrum, and nuclear magnetic resonance spectrum.