Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.
Four mutants of phe V, a gene coding for tRNAPhe in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2+U2 in tRNAPh', is expressed about 10-fold lower in transformed cells than wild-type phel': This mutant, unlike the remaining three (G15+A15, G44+A44, m7G46+A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV( U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)-+T(-6), restores expression of the mutant U2 to about the level of wild-type tRNAPh'.A gene, p h e v coding for tRNAPhe in Escherichia coli, was isolated in this laboratory by virtue of the fact that plasmids or cosmids carrying pheV could complement the thermosensitivity of certain mutants affecting phenylalanyl-tRNA synthetase (PheRS) [l]. Subsequently, mutants in pheV were obtained after mutagenesis of the gene in vitro . Two criteria were employed in screening for mutants of pheV: (a) deattenuation of b-galactosidase synthesis, while under the control of the attenuator region of the pheS, T operon (which codes for PheRS) by means of an operon fusion: (b) loss of the ability to complement the thermosensitivity of a mutant form of PheRS. By this approach mutants affecting four different positions in tRNAPhe were isolated . All four mutants lose the ability of the wild-type tRNA to complement thermosensitive PheRS. Although the mechanism for the complementation by plasmids carrying wild-type phe V is not entirely understood, it probably involves an increase in the activity or stability of the mutant PheRS in the presence of an excess of tRNAphe, maybe associated with an increased K , for tRNA of the mutant enzyme. Loss of complementation by mutations affecting the structural gene for tRNAPhe may thus be due to less efficient transcription or maturation or an altered interaction with PheRS.Here we describe experiments which investigate the mechanism of loss of complementation by the mutant species of tRNAPhe. Furthermore, we show that one of these mutants may be used to isolate promoter mutants in pheV with increased promoter activity. MATERIALS AND METHODS Strains and plasmidsStrains and plasmids have been described in , except for plasmids pPPl5, a tetracycline-resistant derivative of pBR322 with a 350-base-pair insertion which includes a gene for tRNAPhe (phev),  and pUC19 . Strains IBPC 5311 or IBPC 1671 transformed by plasmi...
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 18.104.22.168); nuclease S1 (EC 3.1.30...
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