Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

According to the conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGS^GLAV, IGS^GLV, IGS^lA, IGS^G and IGS^A; while the strain CG021 has the same types of IGSs except lacking IGS^A. Among these five IGS types, IGS^GLAV is the biggest type, including the gene cluster of tRNA^Glu - tRNA^Lys - tRNA^Ala - tRNA^Val; IGS^GLV includes that of tRNA^Glu-tRNA^Lys-tRNA^Val; IGS^AG, tRNA^Ala-tRNA^Glu; IGS^IA, tRNA^Ile-tRNA^Ala; IGS^G, tRNA^Glu and IGS^A, tRNA^Ala. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non-coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Streptomyces cholesterol oxidase was produced in Escherichia coli by a modification of the cholesterol oxidase gene (choA′) in which the native codons for the precursor NH₂-terminal region and the ribosome binding site were substituted for those favored by E. coli. The choA′ gene was expressed under the control of the lac or tac promoter in a multiple copy plasmid vector, although no expression of the native choA gene from Streptomyces was observed in E. coli. E. coli cells carrying the plasmid, pCo117, produced 2-fold more cholesterol oxidase intracellularly during 18-h culture than did the producing strain of Streptomyces sp. SA-COO cultured for 4 d. The NH₂-terminal amino acid sequence of cholesterol oxidase produced by E. coli appeared to be processed between Ala²⁰ and Ala²¹ of the precursor enzyme, while the Streptomyces enzyme was processed between Ala⁴² and Asp⁴³. Based on the facts that the cholesterol oxidase was stable, could be assayed rapidly, and no endogenous cholesterol oxidase activity was found in any enteric bacteria, we developed two widely applicable, new promoter-probe vectors posessing the choA′ gene, multiple cloning sites, and either a low or high copy number plasmid. Since these plasmids can replicate in enteric bacteria, the new plasmid vectors have a great potential for use in enteric bacteria without the isolation of Cho⁻ mutants.     

Half molecules of serine-specific transfer ribonucleic acids from yeast

The preparation and analysis of half molecules from tRNA^Ser are described. Two pG-halves were isolated which differed only in the presence or absence of an acetyl group on the cytidylic acid residue at position 12. The CCA-half derived from tRNA₁^Ser was isolated pure, while the CCA-half derived from tRNA₂^Ser was isolated as a mixture with the CCA-half from tRNA₁^Ser from which the terminal CpCpA had been cleaved off.The acceptor activity of the combined complementary half molecules was 90% of the one of intact tRNA^Ser. The Michaelis constant and maximal velocity of amino-acylation were found to be identical for tRNA^Ser and the combined fragments.When half molecules were present at different ratios in aminoacylation studies it was found that one pG-half molecule can mediate the charging of several CCA-half molecules. There are indications that the CCA-half molecule alone can accept some serine. The CCA-half molecule alone can be aminoacylated to a rather high degree in the presence of an excess of tRNA_ox^Ser or tRNA^Ser-a and to a small degree in the presence of tRNA_ox^Ala (yeast) but not at all in the presence of tRNA_ox^Phe or tRNA_ox^Val (E. coli).Combinations of half molecules from tRNA^Ser with the opposite half molecules from tRNA^Phe could not be aminoacylated with Ser or Phe or 15 other amino acids although one of the combinations was well associated according to gel electrophoresis and differential melting curves.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

Normal and Mutant Glycine Transfer RNAs

THE glycine-specific tRNAs of E. coli can be grouped into three subspecies which are separated by chromatography on benzoylated DEAE cellulose (BDC): tRNA^Gly₁ (GGG), tRNA^Gly₂ (GGA/G) and tRNA^Gly₃ (GGU/C)^1,2. The tRNA^Gly₁ and tRNA^Gly₂ are specified by the genes, glyU and glyT, respectively, which have been located at 55 and 77 minutes on the E. coli chromosome. Suppressors of tryptophan A gene (trpA) missense mutations and partial diploid strains have been used extensively to characterize the glycine tRNA structural genes (Table 1)^1–3. A common property of these suppressor mutations is that the altered tRNA^Gly is no longer aminoacylated at the normal rate by the glycyl tRNA synthetase (GRS). When ordinary loading conditions are used virtually none of the suppressor tRNA species are amino-acylated. These studies have shown that single gene copies are normally present at the glyT and glyU loci.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

Functional Interaction Between Release Factor One and P-site Peptidyl-tRNA on the Ribosome

Translation termination at UAG is influenced by the nature of the 5′ flanking codon inEscherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA⁺) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA₃^ProversustRNA₂^Pro, tRNA₄^ThrversustRNA_1,3^Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA₂^Gly) and GGG (tRNA_2,3^Gly) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High α-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.     

A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 × 10⁻⁵ to a high of 6.9 × 10⁻⁴ per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.     

Inverse PCR-Based Method for Isolating Novel SINEs from Genome

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA^Ala molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA^Ala-derived region at the 5′ end, a tRNA-unrelated region, and AT-rich region at the 3′ end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA^Ala gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA^Ala. The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.     

Biological function of modified nucleotides T54 and Ψ55 of yeast tRNAAla

The 3′ half molecule of yeast tRNA^Ala (nucleotides 36–75) was hybridized with a DNA fragment (5′GGAATCGAACC 3′) and the hybrid was then digested withE. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3′ half molecule at the 3′ side of nucleotide Ψ₅₅, thus a fragment C₃₆-Ψ₅₅ was prepared. The 3′-terminal T or TΨ of this fragment was removed by one or two cycles of periodate oxidation and β-elimination. The products were fragments C₃₆-T₅₄ and C₃₆-G₅₃. Three yeast tRNA_Ala fragments C₅₆-A₇₆, U₅₅-A₇₆ (with Ψ₅₅ replaced by U), U₅₄-A₇₆ (with T₅₄Ψ₅₅ replaced by UU) were synthesized and ligated with three prepared fragments (C₃₆-Ψ₅₅, C₃₆-T₅₄ and C₃₆-G₅₃) respectively by T4 RNA ligase. The products were further ligated with the 5′ half molecule (nu-cleotides 1–35). Using this method, one reconstituted yeast tRNA^Ala (tRNAr) and two yeast tRNA^ALa analogs: (i) tRNAa with U₅₅ instead of Ψ₅₅; (ii) tRNAb with U₅₄U₅₅ instead of T₅₄Ψ₅₅ were synthesized. The charging and incorporation activities of these three tRNAs were determined. In comparison with the reconstituted tRNA, the charging activity was 75% for tRNAa and 45% for tRNAb and the incorporation activity was 65% for tRNAa and 70% for tRNAb. These results suggest that the modified nucleotides T₅₄ and Ψ₅₅ play an important role in yeast tRNA^Ala function.     

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-N⁶-(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.