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Numerous misacylations occur on heterologous systems containing unfractionated tRNAs from yeast or from Bacillus stearothermophilus and pure valyl-tRNA synthetase from B. stearothermophilus or from yeast, when special aminoacylation conditions are used. In the homologous system and in heterologous systems where the unfractionated tRNAs and the enzyme originate from prokaryotic organisms (B. stearothermophilus and Escherichia coli), the errors are seldom. This phenomenon is explained by competition effects between the cognate tRNAVal and non-cognate tRNAs for the valyl-tRNA synthetase. But using pure tRNA species, errors can be observed in such systems, even under classical assay conditions; in particular it was shown that the valyl-tRNA synthetase from B. stearothermophilus catalyses the misacylation of E. coli tRNAIle and tRNAtMet and of yeast tRNAtMet and tRNAPhe. These reactions are characterized by Km values slightly increased as compared to the value obtained in the cognate system and by V values decreased by a factor of about 40 to 3000 compared to the cognate tRNA species. In the presence of dimethylsulfoxide, the rate and the extent of those misacylation reactions are enhanced. In the case of E. coli tRNAPhe, the misacylation occurs only in the presence of the organic solvent. In no case however, new aminoacylation errors are induced at high temperature (50–75 °C) in the presence of the thermostable valyl-tRNA synthetase from B. stearothermophilus; only an increase of the rates of the aminoacylation reactions which already occur at 30 °C has been observed at higher temperature. Thus organic solvent and heat must have distinct effects on the essential parameters determining the specificity of the tRNA aminoacylation reactions. Also it has been observed that the most easily misacylated tRNA species by valyl-tRNA synthetase from B. stearothermophilus are the same as those which are misacylated by the valyl-tRNA synthetases from E. coli and from yeast. This observation suggests the existence of a family of tRNAs containing besides of tRNAVal, other tRNAs such as tRNAIle, tRNAtMet and tRNAPhe, and which are likely to be related from a phylogenic point of view. Moreover these tRNA species have also been found to be easily misacylated by other aminoacyl-tRNA synthetases namely the enzymes specific for isoleucine and phenylalanine, thus suggesting more generally the existence of interacting tRNA-aminoacyl-tRNA synthetase families.

The effect of N-[9-(β-d-ribofuranosyl) purin-6-ylcarbamoyl]threonine (t6A) adjacent to anticodon U*-C-U of yeast tRNAArgIII (where U* is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide-directed binding of tRNA on ribosomes, (b) the ribosome-free trinucleotide binding to the anticodon. (c) the anticodon-anticodon binding test. The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNAArgIII with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A-G-A triplet, and (c) E.coli tRNAServ (anticodon G-G-A). We therefore conclude that the stabilization effect of t6A occurs on U · A and U · G base pairs adjacent to the 5′ side of the modified nucleoside, most probably by stacking.

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in beta-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function.

In this paper we report on the properties of the purified isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110000. The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity at 65 °C for the isoleucine-dependent ATP-PPi exchange reaction and at 45 °C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein. An association constant of 0.11 nM−1 was determined at 70.5 °C. Formation of this tRNA · enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM−1 measured. The same results were obtained either with tRNA from Escherichia coli or B. stearothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3′-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA · enzyme complex has been demonstrated. The intermediate isoleucine · AMP · enzyme complex with a molar ratio of 1:1:1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA · enzyme complex. Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine · AMP · enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex.

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase., info:eu-repo/semantics/published

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments (‘half molecules’) derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5′ and 3′ tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: (a) the stability of these eubacterial tRNA variants in the eukaryotic oocytes; (b) the enzymatic modification of the adenosine at position 37 (3′ adjacent to the anticodon) and (c) aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-β-d-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.