tRNA synthesis in germinating pollen

The synthesis of tRNA was demonstrated in pollen ofNicotiana tabacum L. according to the incorporation of labeled uracil, adenosine and guanosine during 4 h of germination. tRNA was extracted from postribosomal supernatant and purified by polyacrylamide gel electrophoresis. The incorporation of guanosine was about 1.68 times higher than that of adenosine. This finding indicates that the whole tRNA chain is formed in pollen tubes.     

Enzymatic synthesis of tRNA fragments

In the present paper the results of enzymatic synthesis of yeast tRNA1Val fragments have been summarized. It is shown that complex use of nucleolytic enzymes is a convenient and effective method of synthesis of the defined sequence oligoribonucleotides. The consecutive use of different nucleolytic enzymes (ribonucleases with different substrate specificity and polynucleotide phosphorylase) and RNA ligase has permitted to obtain various fragments (or their analogs) of T psi-loop, D-arm, anticodon arm and acceptor stem. Some fragments containing modified nucleosides such as tetranucleotide GpDpCpGp (fragment 15-18), octanucleotide GpUpCpUpApGpDpC (analog of fragment 10-17), nonanucleotide GpTpUpCpGpApUpCpC (analog of T psi-loop), decanucleotide psi pCpUpGpCpUpUpIpApC (analog of fragment 27-36), hexanucleotide CpApCpGpCpA (fragment 36-41) and others were synthesized.     

Labile protecting groups in tRNA synthesis

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.     

Translational control of protein synthesis by tRNA unrelated to changes in tRNA concentration

Summary The rate of translation of mRNA into protein is controlled, at least in part, by the concentration of tRNA. However, as one tRNA molecule can recognize more than one codon, different codons translated by the same tRNA may be translated at different rates. The difference in response of a tRNA molecule to different codons could also regulate the rate of protein synthesis. Evidence for this theory is presented, and Crick's wobble hypothesis is extended to include the relative strengths of different codon-anticodon interactions.     

Protein synthesis in Escherichia coli with mischarged tRNA

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.     

Functional differences in protein synthesis between rat liver tRNA and tRNA from Novikoff hepatoma.

Synthesis of ovalbumin in fragmented oviduct magnum explants of immature, estrogen-stimulated chicks has been studied in the presence of exogenous tRNA. tRAN from Novikoff hepatoma specifically inhibited ovalbumin synthesis, determined by precipitation with antisera. In addition, the major protein(s) synthesized in the presence of hepatoma tRNA had higher electrophoretic mobility than ovalbumin, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. tRNAs from rat liver, rooster liver, and hen oviduct did not affect ovalbumin synthesis, although oviduct tRNA is stimulatory during the earlier stages of estrogen stimulation.     

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.     

Modulation of lambda integrase synthesis by rare arginine tRNA

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.     

Isoleucyl initiator tRNA does not initiate eucaryotic protein synthesis

Initiator tRNA from yeast (tRNAMeti) was quantitatively misaminoacylated with L-isoleucine using isoleucyl-tRNA synthetase from Escherichia coli. Surprisingly the misaminoacylated Ile-tRNAMeti neither participates in nor inhibits the initiation of globin synthesis in a rabbit reticulocyte lysate, whereas Met-tRNAMeti readily initiates protein synthesis in the same system. The incompetent behavior of Ile-tRNAMeti may be related to the observation that in vitro it does not form a stable complex with eucaryotic initiation factor 2 (eIF-2) and GTP, under conditions which lead to a stable eIF-2 X GTP X Met-tRNAMeti ternary complex. This indicates that eIF-2 can discriminate between the side chains of the aminoacyl adducts of the tRNAMeti during ternary complex formation, the first essential step in initiation of eucaryotic protein synthesis.     

Temperature sensitive mutants of Escherichia coli for tRNA synthesis

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3⁺-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA^Tyr was demonstrated. Electrophoretic fractionation of ³²P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions.     

Increased levels of glycine tRNA associated with collagen synthesis

Analysis of codon usage for chick Type I collagen indicates that 89% of glycine codons are GGU/C. Since collagens are one-third glycine, chick Type I collagen synthesis should require large amounts of tRNAGly with the anticodon GCC. Earlier chromatographic studies of chick tRNA had indicated that connective tissues showed altered tRNAGly isoacceptor profiles [P. J. Christner and J. Rosenbloom (1976) Arch. Biochem. Biophys. 172, 399-409; H. J. Drabkin and L. N. Lukens (1978) J. Biol. Chem. 253, 6233-6241]. We have therefore used both two-dimensional gel electrophoresis and hybridization analysis to investigate whether collagen synthesis in chick connective tissues is associated with expression of a novel tRNAGly. Liver and calvaria tRNAs produced qualitatively similar patterns when separated on 2-D gels. Northern blots of 2-D-separated tRNAs from liver and calvaria, when hybridized to genes for vertebrate tRNAGly isoacceptors with GCC or UCC anticodons, showed hybridization to the same tRNAs in both tissues. Quantitation of tRNA species by dot blot hybridization indicated an increase in levels of the tRNAGly isoacceptor with anticodon GCC. Tissues synthesizing Type I collagen had a two- to threefold increase in this tRNA while tissues synthesizing Type II collagen showed a more modest increase. We conclude that elevated tRNAGly levels associated with collagen synthesis are due to increased amounts of the same isoacceptor which is the major tRNAGly in other tissues.     

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Background  

Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.     

A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Here we describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.     

Preferential usage of tRNA isoaccepting species in collagen synthesis.

A new double label technique is described for determining the usage of individual isoaccepting tRNA species by in vitro protein-synthesizing systems. Employing this method we show that of four major species of glycyl-tRNA one which is cognate to GGU and GGC is used predominantly in collagen synthesis by polysomes isolated from embryonic chick calvaria. A similar preferential usage was found for one of two alanyl-tRNA species. No preference for any particular isoaccepting tRNA species was observed in the synthesis of noncollagenous proteins by either calvaria or liver polysomes except for the disproportionate usage of one lysyl-tRNA species. Whether such preferential usage permits translational control of collagen synthesis in vivo remains to be determined.