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. I n 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 tRNAVa1 and noncognate 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 tRNA,Met and of yeast tRNA,Met and tRNAPhe. These reactions are characterized by K , 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. I n the presence of dimethylsulfoxide, the rate and the extent of those misacylation reactions are enhanced. I n the case of E . coli tRNAPhe, the misacylation occurs only in the presence of the organic solvent. I n no case however, new aminoacylation errors are induced a t 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 a t 30 "C has been observed a t 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 valyltRNA synthetases from E . coli and from yeast. This observation suggests the existence of a family of tRNAs containing besides of tRNAVB', other tRNAs such as tRNATle, tRNAyet 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 possibility of the tRNAs to be enzymatically mischarged with a wrong amino acid seems to be a rather general feature as numerous incorrect aminoacylation systems have now been described [i -31 (and references therein). These mischarging reactions have especially been detected in vitro, under various experimental conditions, either in heterologous systems where the enzyme and the tRNA came from different sources, but also in homologous systems, where one pure tRNA species interacts Enzyme. Valyl-tRNA synthetase (EC 184.108.40.206). Eur. J. Biochem. 45 (1974)with one non-cognate pure ...
We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.
An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.
Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.
We have constructed eight anticodon-modified Escherichia coli initiator methionine (Met) 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-~-~-ribofuranosylpurine-6-yl)carbamoy~]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.Some time ago a correlation between the anticodon sequence of a tRNA and the identity of neighbouring modified nucleosides was noted [l] (reviewed in ). Thus, the hypermodified nucleoside t6A, N-[N-(9-P-~-ribofuranosylpurine-6-yl)carbamoyl]threonine, or a derivative thereof is located in position 37 of tRNAs having anticodons terminating in a uridine. One enigmatic exception is the initiator methionine tRNA of Escherichia coli, for most other tRNAs including the eubacterial elongator Met-tRNA, which has the same anticodon sequence, and the eukaryotic initiator tRNA contain the A-37 modification .To study the effect of structural modifications in the anticodon loop on the modification of A-37, we have turned to recombinant RNA methods based on T4 RNA ligase. These techniques are particularly well-suited to the preparation of related tRNA chimera, which have substitutions in or near the anticodon, since fragments serving as starting material for the tRNA variant can be readily obtained from controlled
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 /?-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.Several lines of evidence now suggest that the overall functional efficiency of a tRNA molecule in protein synthesis is dependent on the structure of the whole of the molecule and its relation to the anticodon. Thus Gorini, observing the varying efficiencies of different nonsense suppressors remarked [I] "it appears that a 'correct' anticodon obtained by mutation can be out of context in the resulting tRNA suppressor molecule". In addition to the occurrence of invariant nucleotides in the tRNA molecule, it is known that base usage in other positions is often far from random . One of the most versatile ways of investigating the importance of particular bases or regions of the molecule is to construct tRNAs with modified primary structure. This may be done either by modification of the DNA or the RNA sequence. Temple et al. have recently reported the construction of an active suppressor tRNALy" by site-directed mutagenesis of the gene . By modification of the RNA sequence, Bruce et al. have succeeded in synthesising a biologically active amber suppressor by modification at the RNA level of yeast tRNAPhe . Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe , tRNAASp  and tRNAkg .Essentially three reasons led us to select yeast tRNACy" as an interesting species for modification. Among the anticodon changes that may be easily introduced, one should lead to a UGA suppressor species, the activity of which may be readily assayed in vivo as well as in vitro, The normal tRNACYs anticodon, GCA, is conveniently split by ribonuclease TI. Finally, the availability of high specific activity [35S]cysteine should facilitate characterisation of polypeptides synthesized in vivo or in vitro.Enzymes. RNAse TI (EC 220.127.116.11); nuclease S1 (EC 3.1.30...
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