Polarization-controlled tunable directional spin-driven photocurrents in a magnetic metamaterial with threefold rotational symmetry.

Future spintronics and quantum technologies will require a portfolio of techniques for manipulating electron spins in functional nanodevices. Especially, the establishment of the methods to control spin current is the key ingredient essential for the transfer and processing of information, enabling faster and low-energy operation. However, a universal method for manipulating spin currents with full-directional controllability and tunable magnitude has not been established. Here we show that an artificial material called a magnetic metamaterial (MM), which possesses a novel spintronic functionality not exhibited by the original substance, generates photo-driven ultrafast spin currents at room temperature via the magneto-photogalvanic effect. By tuning the polarization state of the excitation light, these spin currents can be directed with tunable magnitude along an arbitrary direction in the two-dimensional plane of the MM. This new concept may guide the design and creation of artificially engineered opto-spintronic functionalities beyond the limitations of conventional material science.

Chirality-Dependent Circular Photogalvanic Effect in Enantiomorphic 2D Organic-Inorganic Hybrid Perovskites.

The control of the optoelectronic properties of 2D organic-inorganic hybrid perovskite (2D-OIHP) lead halides is an increasingly prevalent topic. Herein, the observation of the circular photogalvanic effect (CPGE) in new enantiomorphic 2D-OIHP lead iodides is reported, which are synthesized as a first OIHP-related system belonging to a chiral space group by incorporating organic chiral cations into the inorganic layers of lead iodides. The CPGE is an optoelectronic phenomenon associated with the spin-orbit coupling of heavy atoms in noncentrosymmetric systems. Owing to the CPGE, light-helicity-dependent steady photocurrents are generated without an external bias voltage under the irradiation of circularly polarized light. Furthermore, the sign reversal of the CPGE photocurrent depending on the chirality of the designed 2D-OIHP lead iodides is observed. This result indicates formation of the theoretically predicted radial spin-polarized texture in k-space of chiral systems owing to spin-momentum locking. Hence, chiral 2D-OIHP lead halides can be a promising platform for engineering opto-spintronic functionalities.

Phosphoryl oligosaccharides of calcium enhance mineral availability and fluorapatite formation.

OBJECTIVES: Phosphoryl oligosaccharides of calcium (POs-Ca) are a highly soluble calcium source and can keep the solubility of calcium and fluoride ions. The aim of this study was to investigate the effect of calcium (from POs-Ca) and fluoride ions penetrate into subsurface enamel lesions in vitro. DESIGN: Demineralized bovine enamel slabs were remineralizedin vitro for 24 h at 37 °C with artificial saliva (AS) containing POs-Ca and various fluoride concentrations (0-100 ppm), or AS containing different levels of POs-Ca adjusted to a Ca/P ratio of 0.4-3.0 and fluoride, then were analyzed using Transversal microradiography. From those results, remineralization effects with optimal conditions were compared between POs-Ca and calcium chloride (CaCl2). To determine the form of incorporated fluoride, we analyzed the chemical state and local structure of fluorine atoms integrated into enamel subsurface lesions using micro X-ray absorption near-edge structure (µ-XANES) spectroscopy. RESULTS: A significant mineral recovery rate was observed with POs-Ca and fluoride at 0.5 or 1.0 ppm (n = 6, p < 0.05), as well as a Ca/P molar ratio of 1.67 (n = 5, p < 0.05). Under those conditions, the mineral recovery rate of AS containing POs-Ca (37.9 ± 7.3%) was significantly greater than that of CaCl2 (15.0 ± 9.6%) (n = 5, mean ± SD, p < 0.05). µ-XANES spectra analysis of the samples indicated that the dominant form of fluorine atoms in enamel subsurface lesions was fluorapatite. CONCLUSIONS: POs-Ca with fluoride-derived diffusion into subsurface enamel lesions facilitated formation of fluorapatite phases.

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Optimization of calcium concentration of saliva with phosphoryl oligosaccharides of calcium (POs-Ca) for enamel remineralization in vitro.

OBJECTIVE: Phosphoryl oligosaccharides of calcium (POs-Ca) are highly soluble calcium source made from potato starch. The aim of this study was to investigate the optimal concentrations of POs-Ca for the remineralization of subsurface enamel lesions in vitro. DESIGN: Demineralized bovine enamel slabs (n=5) were remineralized in vitro for 24h at 37°C with artificial saliva (AS) containing 0-0.74% POs-Ca to adjust the Ca/P ratio to 0.4-3.0, then sectioned and analysed by transversal microradiography (TMR). The data were analysed by Scheffe's post hoc test. The Ca/P ratio with most remineralization was used to investigate the effect of calcium on enamel remineralization (n=11). The demineralized slabs were treated with AS with calcium-chloride- (CaCl2-) or POs-Ca with an identical calcium content, and sectioned for TMR and wide-angle X-ray diffraction (WAXRD) analyses to evaluate the local changes in hydroxyapatite (HAp) crystal content. The data were analysed using the Mann-Whitney U-test. RESULTS: The highest mineral recovery rate resulted from addition of POs-Ca to adjust the Ca/P to 1.67. At this ratio, the mineral recovery rate for AS containing POs-Ca (24.2±7.4%) was significantly higher than that for AS containing CaCl2 (12.5±11.3%) (mean±SD, p<0.05). The recovery rate of HAp crystallites for AS containing POs-Ca (35.7±10.9%) was also significantly higher than that for AS containing CaCl2 (23.1±13.5%) (p<0.05). The restored crystallites were oriented in the same directions as in sound enamel. CONCLUSIONS: POs-Ca effectively enhances enamel remineralization with ordered HAp at a Ca/P ratio of 1.67.

Genetic-code evolution for protein synthesis with non-natural amino acids.

The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.

Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography.

We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase.

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, L-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA(Pyl)). The PylRS-tRNA(Pyl) pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N(epsilon)-(tert-butoxycarbonyl)-L-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the alpha-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain alpha-groups: alpha-hydroxyacid, non-alpha-amino-carboxylic acid, N(alpha)-methyl-amino acid, and D-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA(Pyl) in E. coli in the presence of the alpha-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA(Pyl) pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.

Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.

Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNA(Pyl). In this study, N(epsilon)-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N(epsilon)-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA(Pyl) by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an N(epsilon)-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for N(epsilon)-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing N(epsilon)-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.

Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog.

To guarantee specific tRNA and amino acid pairing, several aminoacyl-tRNA synthetases correct aminoacylation errors by deacylating or "editing" misaminoacylated tRNA. A previously developed variant of Escherichia coli tyrosyl-tRNA synthetase (iodoTyrRS) esterifies or "charges" tRNA(Tyr) with a nonnatural amino acid, 3-iodo-l-tyrosine, and with l-tyrosine less efficiently. In the present study, the editing domain of phenylalanyl-tRNA synthetase (PheRS) was transplanted into iodoTyrRS to edit tyrosyl-tRNA(Tyr) and thereby improve the overall specificity for 3-iodo-l-tyrosine. The beta-subunit fragments of the PheRSs from Pyrococcus horikoshii and two bacteria were tested for editing activity. The isolated B3/4 editing domain of the archaeal PheRS, which was exogenously added to the tyrosylation reaction with iodoTyrRS, efficiently reduced the production of tyrosyl-tRNA(Tyr). In addition, the transplantation of this domain into iodoTyrRS at the N terminus prevented tyrosyl-tRNA(Tyr) production most strongly among the tested fragments. We next transplanted this archaeal B3/4 editing domain into iodoTyrRS at several internal positions. Transplantation into the connective polypeptide in the Rossmann-fold domain generated a variant that efficiently charges tRNA(Tyr) with 3-iodo-l-tyrosine, but hardly produces tyrosyl-tRNA(Tyr). This variant, iodoTyrRS-ed, was used, together with an amber suppressor derived from tRNA(Tyr), in a wheat germ cell-free translation system and incorporated 3-iodo-l-tyrosine, but not l-tyrosine, in response to the amber codon. Thus, the editing-domain transplantation achieved unambiguous pairing between the tRNA and the nonnatural amino acid in an expanded genetic code.

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases.

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA(Pyl) pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA(Pyl) in Chinese hamster ovary cells. When tRNA(Pyl) was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA(Pyl) pair facilitated the most efficient incorporation of a pyrrolysine analog, N(epsilon)-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA(Pyl) system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N(epsilon)-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N(epsilon)-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.

Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase.

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the "22nd" genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA(Pyl) with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the "open" states to the "closed" states upon AMPPNP binding. On the other hand, the beta 7-beta 8 hairpin exhibits multiple conformational states, the open, intermediate (beta 7-open/beta 8-open and beta 7-closed/beta 8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the beta 7-beta 8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the beta 7-beta 8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA(Pyl). Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and alpha-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.

Crystal structures of tyrosyl-tRNA synthetases from Archaea.

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid.

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.

Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion.

The genetic code in a eukaryotic system has been expanded by the engineering of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo-L-tyrosine rather than L-tyrosine. In the present study, we determined the 3-iodo-L-tyrosine- and L-tyrosine-bound structures of the 37V195C mutant of the E. coli TyrRS catalytic domain at 2.0-A resolution. The gamma-methyl group of Val-37 and the sulfur atom of Cys-195 make van der Waals contacts with the iodine atom of 3-iodo-L-tyrosine. The Val-37 and Cys-195 side chains are rigidly fixed by the neighboring residues forming the hydrophobic core of the TyrRS. The major roles of the two mutations are different for the 3-iodo-L-tyrosine-selective recognition in the first step of the aminoacylation reaction (the amino acid activation step): the Y37V mutation eliminates the fatal steric repulsion with the iodine atom, and the Q195C mutation reduces the L-tyrosine misrecognition. The structure of the 37V195C mutant TyrRS complexed with an L-tyrosyladenylate analogue was also solved, indicating that the 3-iodo-L-tyrosine and L-tyrosine side chains are similarly discriminated in the second step (the aminoacyl transfer step). These results demonstrate that the amino acid-binding pocket on the 37V195C mutant is optimized for specific 3-iodo-L-tyrosine recognition.

Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase.

Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the amino acid activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the amino acid activation step in the aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the amino acid activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the amino acid activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the aminoacyl transfer reaction.

The Escherichia coli argU10(Ts) phenotype is caused by a reduction in the cellular level of the argU tRNA for the rare codons AGA and AGG.

The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNA(Arg) species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30 degrees C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43 degrees C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30 degrees C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43 degrees C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.