Takashi Yokogawa

Cell-free translation reconstituted with purified components.

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

An unnatural base pair for incorporating amino acid analogs into proteins

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography–mass spectrometry (LC-MS) analysis of the products. This coupled transcription–translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea

A modified base at the first (wobble) position of some tRNA anticodons is critical for deciphering the genetic code. In eukaryotes and eubacteria, AUA codons are decoded by tRNAsIle with modified bases pseudouridine (and/or inosine) and lysidine, respectively. The mechanism by which archaeal species translate AUA codons is unclear. We describe a polyamine-conjugated modified base, 2-agmatinylcytidine (agm(2)C or agmatidine), at the wobble position of archaeal tRNA(Ile) that decodes AUA codons specifically. We demonstrate that archaeal cells use agmatine to synthesize agm(2)C of tRNA(Ile). We also identified a new enzyme, tRNA(Ile)-agm(2)C synthetase (TiaS), that catalyzes agm(2)C formation in the presence of agmatine and ATP. Although agm(2)C is chemically similar to lysidine, TiaS constitutes a distinct class of enzyme from tRNA(Ile)-lysidine synthetase (TilS), suggesting that the decoding systems evolved convergently across domains.

Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system

Mitochondrial (mt) tRNAMet has the unusual modified nucleotide 5-formylcytidine (f5C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNAMet carrying f5C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNAMet lacking f5C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNAMetm with the anticodon ac4CAU (ac4C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNAMet (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f5C modification in mt tRNAMet plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f5C-G and f5C-A based on the chemical properties of f5C are presented.

Existence of nuclear-encoded 5S-rRNA in bovine mitochondria.

A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear‐encoded 5S RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5S RNA is actually located inside the mitochondrial inner membrane. The 5S rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) 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 tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 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.

Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria

Background Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti‐codon stem. It has been a long‐standing problem to prove experimentally how these tRNAsSer work in the mt translation system.

Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts

We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA•oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.

The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons

The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt. In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.

Conformatzonal Properties of a Novel Modified Nucleoside, 5-Formylcytidine, Found at the First Position of the Anticodon of Bovine Mitochondrial tRNAMet

Abstract Conformational properties of a novel modified nucleoside, 5-formylcytidine (f5C), which is found at the first position of the anticodon of bovine mitochondrial tRNAMet, were analyzed by 1H-NMR spectroscopy. f5C has a normal amino tautomeric form at position 4 of the base moiety. The results indicate the presence of an intramolecular hydrogen bond between the carbony1 of the 5-formyl group and the 4-amino function. f5C was found to exhibit the C3′-endo conformation exclusively and the enthalpy difference (ΔH) between the C2′-endo and C3′- endo forms was found to be 1.56 ± 0.13 kcal/mol, indicating f5C to be one of the most conformationally rigid nucleosides yet analyzed. The conformational rigidity of f5C may contribute to regulation of codon recognition by tRNAMet.